Converging Biochemical Pathways in Psychiatric Disorders
by
Takahiro Soda
B.S., University of California at Los Angeles (UCLA), (2005)
Submitted to the Department of Brain and Cognitive Sciences
ARCHIVES
In Partial Fulfillment of the Requirements of the Degree of
IMASSACHUSETIS
Doctor of Philosophy in the field of
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Neuroscience
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MASSACHUSETTS INSTITUTE OF TECHNOLOGY
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June 2012
@2012 Massachusetts Institute of Technology. All rights reserved
Signature of Author..................................
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Department of Brain and Cognitive Sciences
May 30, 2012
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Li-Huei Tsai
Picower Professor of Neuroscience,
Department of Brain and Cognitive Sciences
Director, theP/ower Institute for Learning and Memory
Accepted by ....................
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Earl K. Miller
Picower Professor of Neuroscience
Director, BCS Graduate Program
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Converging Biochemical Pathways in Psychiatric Disorders
by
Takahiro Soda
Submitted to the Department of Brain and Cognitive Sciences
on June 1, 2012 in partial fulfillment of the requirements for the Degree of
Doctor of Philosophy in the field of
Neuroscience
Abstract:
According to the World Health Organization, neuropsychiatric diseases account for
approximately one third of years lost to disability. Yet, despite this huge disease burden, there is
a lack of new treatments under development: approved treatments all essentially target the
same target(s), if the target itself is known. There is now considerable evidence for a common
set of heritable risk for psychiatric disorders including schizophrenia, bipolar disorder, as well as
autism. Many of these risk alleles affect genes implicated in neuronal development with known
roles at an early stage; these genes would have an effect on the individual before the onset of
overt symptoms or diagnosis. Furthermore, many of the genes identified are known to
participate in established pathways that are relevant for neuronal development and function. It is
important then to address the causality between these signaling pathways that are important for
neurodevelopment, and the risk of developing neuropsychiatric disorder.
The work presented in this thesis represents two projects that aim to work toward this
goal. The first project pertains to the mechanisms of transcriptional repression by DISC1 on
ATF4-mediated gene transcription. The second project presents some initial steps towards
uncovering the role of BCL9 in neuronal development.
Thesis Supervisor: Li-Huei Tsai
Title: Picower Professor of Neuroscience,
Department of Brain and Cognitive Sciences
Director, the Picower Institute for Learning and Memory
3
Acknowledgments:
First and foremost, I would like to express my utmost and sincere gratitude to my advisor,
Professor Li-Huei Tsai for her continuous patience and support during my Ph.D study and
research. Her drive and dedication is a constant source of inspiration.
I would also like to thank my committee members, Professors Mriganka Sur, Yingxi Lin, and
Guoping Feng, who, despite their busy schedules have taken the time to discuss my findings
and to help put them into context, as well as Professor. Akira Sawa, who has graciously agreed
to travel from Baltimore specifically for my defense.
I would also like to thank my collaborators Yu Park, Dr. Sangki Park, for providing me with
strong evidence to further give me confidence regarding my claims, Dr. Ishizuka for proving me
with key reagents for testing the hypothesis regarding DISC1 PKA phosphorylation, Dr. Aguet
and Dr. Rando for providing us with the BCL9/9L floxed mice. I have been blessed that so many
established researchers have graciously extended their assistance.
I also wish to acknowledge Dr. Christopher Frank for initiating the work regarding the
DISC1/ATF4 interaction, and for his patience and advice throughout the development of the
project.
I would also like to thank the members of the Tsai lab, past and present, that have helped me
both materially, intellectually, and quite possibly, spiritually as I navigated through this process.
In no particular order, I would like to thank the following:
Andre Fisher and Farah Sananbezi, for helping guide me through my first immunoprecipitation
and fear conditioning experiments as well as providing me with excellent saffron with which to
make Chelow.
Lilly Moy, for affirming that it is not abnormal for using a fume hood to prevent having a bubble
underneath a screen protector for the computer, as well as showing me without complete
disdain that 3% BSA works better than 5% milk, in some contexts.
Jodel Giraud and Zhigang Xie for kindly showing me how to do my first cloning projects, and for
being a constant and simultaneous source of levity and knowledge, most often simultaneously
Sangki Park for showing how to do neuronal cultures efficiently, and most importantly for
helping set a paradigm for psychiatric disorder research
Froylan Calderon for teaching me how things are done in Mexico, and how to take excellent and
accurate images on the confocal and epifluorescent microscopes,
Yingwei Mao for showing me the magic behind science, for having many more ideas than he
could ever have time to investigate by himself, being a patient mentor and occasional
contributor to the ridiculous bantor that surrounded our bay, and for countless insights
throughout the development of the project
4
Susan Su, who provided me with PSD preps with which I used up to do preliminary experiments
without having any clue how long the procedure takes,
Karun Singh, for countless conversations regarding science, and many more conversations that
have absolutely nothing to do with anything,
Dinos Meletis, for his excellent sense of time and his patience as he pushed me through the
entirety of the BCL9 flox cloning process,
Xuecai Ge, who helped me tremendously during the yeast-2-hybrid process,
Cillian King, my partner in crime for the initial testing phase of the DISC1/ATF4 interaction,
Ying Zhou, Mali Eichler, Katie Schlieper, for their patience and mouse mating skills, without
which my mouse colonies would have surely gone extinct
Nadine Joseph, Emma Quinn, Ana Rosario, Emily Kilptrick for their excellent demonstration of
button response in response to repeated shear force
Ram for Ice cream runs and insights regarding in vitro experiments
Johannes for telling me about Jasper and teaching me European geography
Martin Kahn for being the hardest working graduate student during his tenure in the lab, despite
being a summer student
Wen-Yuan Wang for his knowledge of promotors and FACs sorting, helping me design a
mutation without inadvertently creating another promotor binding site, and for demonstrating
excellent work-life balance
Matt Dobbin for his patience in teaching me the basics of the confocal machine, and helping me
get rid of Ampicillin plates
Alyssa Baccarella and Omer Durak, for letting me experience two very different kinds of
mentorship relationships, and for helping me achieve my dream of 24 hour continuous lab
bench occupancy,
Paola Giusti-Rodriguez, for being a part of the late shift, without which my productivity in lab
would have dropped even lower,
Neal White, Scott Adams for making sure I really needed everything I ordered, and making sure
I got them when they were delivered, and for keeping everyone in check
Yea Jin Kaeser-Woo, for agreeing to shepherd the BCL9 project to fruition,
Zack Flood and Ester Kwon for breathing new life into the bay,
5
Sandra Siegert for tips on FACs sorting, Elizabeta Gjoneska for an optimized ChIP protocol,
Damien Rei for FACs scheduling, and countless other people that have helped me throughout
my tenure in the lab.
I would also like to thank the members, current and former, of Dr. Michael S. Levine's lab, who
were kind enough to mentor me through my undergraduate years. Dr. Levine, without this
experience I would certainly have not decided to continue to do research. The lab environment
was such that it taught me that research can be producting, rewarding, and fun. I would like to
especially thank Dr's Carlos Cepeda and Nanping Wu, who introduced me to
electrophysiological techniques and allowed me to use their rigs, and gave me advice
throughout my undergraduate years and beyond.
I would also like to thank Dr.'s Kevin Noguchi and Janice Carlson, who were kind enough to
allow me to join them as a 1st year college student to assist in their research despite everything
that they had heard and experienced regarding 1st year undergraduates.
I would also like to thank my classmates and colleagues in the HST entering class of 2005, who
have kept me grounded and motivated throughout the process, as well as my friends outside
the lab, who have always served as a reminder that there is life, outside the lab.
Finally, I would like to thank my family for their continued support and understanding. I realize
that my parents probably did not anticipate that I would plan to be a student for this long as well.
Their unquestioning trust in my choices and unwavering support throughout the process has
been a huge source of both strength and comfort.
6
CHAPTER 1: INTRODUCTION
7
Psychiatric diseases: cost, burden and need for basic research
According to the World Health Organization, neuropsychiatric diseases account for
approximately one third of years lost to disability (YLD) (Table 1). The major depressive
disorders (unipolar and bipolar depression) as well as schizophrenia all separately rank within
the top ten causes of YLD and account for roughly half of the YLD due to neuropsychiatric
causes (Table 2) (source: http://apps.who.int/qhodata/?vid=1 10001 accessed 04/09/2012).
Cause of YLD by Category
% total
YLD
Neuropsychiatric disorders
31.0
Sense organ disorders
14.5
Unintentional injuries
8.1
Infectious and parasitic diseases
8.0
Musculoskeletal diseases
5.0
Respiratory diseases
4.7
Nutritional deficiencies
4.5
Maternal conditions
3.9
Cardiovascular diseases
3.8
Perinatal conditions (e)
3.4
Digestive diseases
2.5
Congenital abnormalities
1.8
Intentional injuries
1.7
Diabetes mellitus
1.6
Oral diseases
1.3
Respiratory infections
1.0
Nutritional/endocrine disorders
1.0
8
Diseases of the genitourinary
system
0.8
Malignant neoplasms
0.7
Skin diseases
0.5
Other neoplasms
0.0
Table 1: Cause of years lost to disability (YLD) as recorded by the World Health
Organization.
Cause, Specific Condition %YLD
Unipolar depressive
disorders
10.9
Refractive errors
4.6
Hearing loss, adult onset
4.6
Other unintentional injuries
4.0
Alcohol use disorders
3.7
Cataracts
3.0
Schizophrenia
2.7
Osteoarthritis
2.6
Bipolar affective disorder
2.4
Iron-deficiency anemia
2.2
Table 2: Top ten causes of years lost to disability as recoraea Dy the woria Hean
Organization.
These statistics highlight the immense burden these disorders place on the human
species as a whole and also the need to better treat the patients that have been identified as
having these debilitating disorders. Yet, despite this huge disease burden, there is a lack of new
treatments under development: approved treatments all essentially target the same target(s), if
9
the target itself is known, investment by the private sector into novel therapeutics for these
disorders have stalled (GlaxoSmithKline PLC, Sanofi-Aventis, AstraZeneca, and Merck, WSJ
http://online.wsi.com/article/SB10001424052748704474804576222463927753954.html
accessed 4/9/2012). The great majority of pharmaceutical companies have halted or refocused
their efforts to other disorders, citing high costs, high probability of failure, and the lack of
promising novel therapeutic targets in the treatment of psychiatric disorders. This places an
even heavier responsibility on basic scientists in academia to identify the molecular
mechanisms underlying these disorders, as the causes for these conditions remain unknown,
and only with a better understanding of the etiology could more drug targets be identified.
The work presented in this thesis pertains to genes with associations to major
depression, bipolar disorder, and schizophrenia. I will briefly introduce the key aspects of each
disorder before going into what is currently known about these disorders, based on the genetic
studies conducted on affected patients, as well as insights gained from research on the
mechanism of action of currently used therapies.
Major depression, bipolar disorder, and schizophrenia
There are many overlapping features between major depression, bipolar disorder, and
schizophrenia, however they can be clinically distinguished by the application of various criteria.
Major recurrent depression is diagnosed based on the presence of a major depressive episode
including the following symptoms nearly every day for a period of at least two weeks, causing
significant distress or dysfunction: depressed mood, loss of interest in enjoying pleasure, loss of
energy, thoughts of worthlessness or guilt, poor concentration, sleep disturbances, change in
appetite/weight, psychomotor retardation/agitation, and thoughts of suicide, with at least one of
the five being either depressed mood or loss of interest or pleasure (American Psychiatric
Association., 2000) (Table 3 ). It is estimated that 8% of Americans have had major depressive
10
episodes, with upwards estimates of prevalence at 11.9% of the population in the United States.
Suicide plays a major role in the risk for suicide, which is the leading cause for death in
adolescents and young adults under the age of 30 (Richards, 2011; Kupfer et al., 2012).
A. The presence of at least one major depressive episode, defined as the presence of five or more of the
following symptoms, present most of the day nearly every day for a minimum of two consecutive weeks.
-Depressed mood*
-Loss of interest or pleasure in most or all activities*
-lnsomnia or hypersomnia
-Change in appetite or weight
-Psychomotor retardation or agitation
-Low energy
-Poor concentration
-Thoughts of worthlessness or guilt
* At least one of these must be present
B. The symptoms do not meet criteria for a mixed episode.
C. The mood disturbance is sufficiently severe to cause marked impairment in occupational functioning,
usual social activities, or relationships with others.
D. The symptoms are not due to the direct physiological effects of a substance (eg, a drug of abuse, a
medication, or other treatment) or a general medical condition (eg, hyperthyroidism).
E. The symptoms are not due to bereavement
Table 3. Diagnostic Criteria for Major Depressive Disorder
Bipolar disorder, though less common, with an estimated 1-3% of the population affected
(Kieseppa et al., 2004; Miller, 2006; Edvardsen et al., 2008; Sachs et al., 2011), nonetheless
has a significant impact on society. The diagnosis of bipolar disorder I is made based on the
presence of a manic or hypomanic episode, which is defined as a period of abnormally and
persistently elevated mood such as distractibility, inflated self-esteem, racing thoughts, a
decreased need for sleep, and risk-taking behavior (Table 4). The presence of a major
depressive episode, while not necessary for diagnosis, is very common: in fact, patients with
bipolar I experience depressive episodes approximately 3 times as often as manic episodes.
Bipolar II is the diagnosis given to patients that experience hypomania and also have at least
one episode of major depression (Benazzi, 2007a). Patients with bipolar 11experience
depressive symptoms as many as 37 times more frequently than symptoms of hypomania (Judd
et al., 2003). A major cause of death in patients with bipolar disorder is suicide, with 15% of
11
patients diagnosed successfully committing suicide (Miller, 2006; Benazzi, 2007b, a). It should
be noted that during episodes of mania, affected individuals could experience delusions, as well
as hallucinations, both of which are characteristics of psychosis. Mood stabilizers such as
lithium and valproic acid are commonly used to treat bipolar disorder, though several recent
reviews of the literature have found that the antipsychotics, which will be mentioned below, are
more effective in the treatment of mania than mood stabilizers (Cruz et al., 2010; Chwieduk and
Scott, 2011; Sachs et al., 2011; Zupancic, 2011).
Diagnostic criteria common to Mania and Hypomania:
A. A distinct period of abnormally and persistently elevated, expansive, or irritable mood, lasting at least 1
week (or any duration if hospitalization is necessary) for mania, and at least 4 days for hypomania.
B. During the period of mood disturbance, three (or more) of the following symptoms have persisted (four
if the mood is only irritable) and have been present to a significant degree:
- Inflated self-esteem or grandiosity
-Decreased need for sleep (eg, feels rested after only 3 hours of sleep)
-More talkative than usual or pressure to keep talking
-Flight of ideas or subjective experience that thoughts are racing
-Distractibility
-Increase in goal-directed activity or psychomotor agitation
-Excessive involvement in pleasurable activities that have a high potential for painful
consequences
E/F. The symptoms are not due to the direct physiological effects of a substance (eg, a drug of abuse, a
medication, or other treatment) or a general medical condition (eg, hyperthyroidism).
Diagnostic criteria specific to mania
C. The symptoms do not meet criteria for a mixed episode.
D.The mood disturbance
1) is sufficiently severe to cause marked impairment in occupational functioning, usual social
activities, or relationships with others,
2) necessitates hospitalization to prevent harm to self or others, or
3) has psychotic features.
Diagnostic criteria specific for hypomania
C. The episode is associated with an unequivocal change in functioning that is uncharacteristic of the
person when not symptomatic.
D. The disturbance in mood and the change in functioning are observable by others.
E. The episode
1)is not severe enough to cause marked impairment in social or occupational functioning,
2) does not necessitate hospitalization, and
3) does not have psychotic features.
Table 4. Diagnostic Criteria for Bipolar Disorder.
Schizophrenia is a chronic debilitating disorder that affects 0.7-3% of the population
(2008). The symptoms of schizophrenia can be broadly put into three categories: positive,
negative, and cognitive. The positive symptoms, most widely associated with schizophrenia, are
12
synonymous with psychosis and include hallucinations, delusions, and disorganized thought.
The negative symptoms are the decrease in, or absence of characteristics such as motivation,
affective response, verbal speech, attention, and enjoyment. People with schizophrenia also
suffer from cognitive impairment including attention, language, memory, and executive function.
Relative cognitive impairment, along with other deficits such as low social function, is often
noted before the onset of positive symptoms, and is seen more often seen in people with
affected siblings. Schizophrenics also exhibit a moderate and appreciable decline in cognitive
function throughout their lifetime. Suicide risk is increased in schizophrenia, particularly amongst
those early in the diagnosis and with high cognitive function, with estimates between 5-10%
successfully committing suicide (Laursen et al., 2012). Even after taking into account the
increased rate of suicide, the diagnosis of schizophrenia is associated with higher mortality; this
can only be partially explained to be due to decreased rate of adherence to medication (Saha et
al., 2007).
A. Two (or more) of the following, each present for a significant portion of time during a 1-month period
(or less if successfully treated)*:
-Delusions
-Hallucinations
-Disorganized speech (eg, frequent derailment or incoherence)
-Grossly disorganized or catatonic behavior
-Negative symptoms, ie, affective flattening, alogia, or avolition
B. For a significant portion of the time since the onset of the disturbance, one or more major areas of
functioning such as work, interpersonal relations, or self-care are markedly below the level achieved prior
to onset (or when the onset is in childhood or adolescence, failure to achieve expected level of
interpersonal, academic, or occupational achievement).
C. Continuous signs of the disturbance persist for at least 6 months.
1). This 6-month period must include at least 1 month of symptoms (or less if successfully
treated) that meet Criterion A, above (ie, active-phase symptoms) and
2). May include periods of prodromal or residual symptoms during which signs of the disturbance
may be manifested by
a). Only negative symptoms or
b). Two or more symptoms listed in Criterion A present in an attenuated form (eg, odd
beliefs, unusual perceptual experiences).
D. Schizoaffective disorder and mood disorder with psychotic features have been ruled out
E. The disturbance is not due to the direct physiological effects of a substance (eg, a drug of abuse, a
medication) or a general medical condition.
F. Relationship to a pervasive developmental disorder
If there is a history of autistic disorder or another pervasive developmental disorder, the additional
diagnosis of schizophrenia is made only if prominent delusions or hallucinations are also present for at
least a month (or less if successfully treated).
13
Table 5. Diagnostic Criteria for Schizophrenia
Therapeutic interventions in place for the treatment of major depression, bipolar disorder,
and schizophrenia
Recent literature reviews have shown a modest, at best, effect of current therapies for
the treatment of severe depression, and no observable effect - beyond placebo- of current
pharmacologic therapies on mild to moderate depression, as defined by a Hamilton Depression
Rating Scale score of less than 25 (Hamilton, 1980). Most major depressive episodes go
undiagnosed (Cepoiu et al., 2008), and most resolve within a matter of months, without
treatment. Current pharmacological therapies used in the treatment of major depressive
disorder (MDD) include the use of monoamine oxidase inhibitors (MAOi's) which act in the
monoamine releasing neurons to inhibit the breakdown of monoamines; tricyclic
antidepressants, which block monoaminergic neurotransmitter reuptake; and more selective
reuptake inhibitors, such as selective serotonin reuptake inhibitors (SSRI's), and serotoninnorepinephrine reuptake inhibitors (SNRI's) (Crupi et al., 2011). The first line therapies for
treatment have become the selective reuptake inhibitors, because of their relatively modest side
effect profile compared to the MAOi's and tricyclics: in fact, MAOi's are now rarely used,
because its use often necessitates dietary restriction and the risk for serotonin syndrome
(Gillman, 2006; Sun-Edelstein et al., 2008). However, even the first line therapies come with a
number of side effects, including drowsiness, sexual dysfunction, weight gain, insomnia, and
anxiety, which are seen in over 10% of patients taking these medications. That all these
medications are effective suggests that all of these neurotransmitter systems play a role in
affect and mechanisms downstream of each of these signaling cascades may play a role in the
manifestation and treatment of MDD, which will be discussed later.
14
The reuptake inhibitors all take about a week before improvements in mood can be
discerned from placebo (Racagni and Popoli, 2010), and the current theory of this delay in
efficacy is due to the requirement of a secondary effect that takes place after the drugs have
reached its site of action. Two potential secondary effects have been presented, which are
neither mutually exclusive nor comprehensive in their explanations. Several animal model
studies (Overstreet et al., 2003; Richardson-Jones et al., 2010) and numerous human brain
imaging studies (Hirvonen et al., 2008; Saijo et al., 2010) have implicated the role of presynaptic
autoreceptors, 5-HT1A in particular for serotonergic neurons of the dorsal raphe (Serretti et al.,
2004) and both 5HT1A and alpha-2-adrenergic receptors in adrenergic neurons of the locus
coeruleus (Berrocoso and Mico, 2007; Ortega et al., 2010), in the delay of onset of
antidepressants. The initial blockade of reuptake triggers an increase in extrasynaptic levels of
the monoamine neurotransmitters, resulting in an activation of autoreceptors at the presynaptic
cell soma of the monoamine-releasing neurons. The activation of autoreceptors at the soma
results in a reduced rate of action potentials in these neurons, which leads to a compensatory
reduction in the level of released neurotransmitter at their postsynaptic sites of action: it is only
with downregulation of these autoreceptors that a functional increase in monoamine
neurotransmitter can be observed (Richardson-Jones et al., 2010). This observation has led to
an interest in using a 5HT1A antagonist in conjunction with SSRI's to improve clinical efficacy or
abridging the time of antidepressant effect. Some trials have shown promising results (Whale et
al., 2010; Portella et al., 2011), while others have shown mixed results (Scorza et al., 2011).
A second reason that has been proposed for the delay in action of reuptake inhibitors, is
that the increase in monoamine levels leads to a cascade of effects postsynaptically that take
weeks to manifest. Some of the effects seen after chronic antidepressant treatment that are
hard to appreciate immediately include things such as increase in dendritic arborization in
numerous brain regions (Alves et al., 2002; Jones et al., 2009), changes in gene transcription of
15
numerous factors that have been implicated in cell survival (Mannari et al., 2008), chronic stress
response(Aubry et al., 1999; Anacker et al., 2011), metabolism (Duman et al., 2009), and
proliferation (Malberg et al., 2000; Santarelli et al., 2003)). I will group these effects and talk
about them as effects on neurons that are already present and established in the neural circuitry
underlying depressive disorders, and effects on newly formed cells that become incorporated
into the neural circuitry underlying depressive disorders.
Effects on the generation of neurons that become incorporated into circuitry
The subgranular zone of the dentate gyrus and subventricular zone have been
established as two areas in which neurogenesis continues into adulthood (Ma et al., 2009). In
the rodent brain, 6% of the neurons in the dentate gyrus are estimated to be new neurons
generated within a month (Cameron and McKay, 1998). While a significantly smaller percentage
is estimated to generated in humans (Amrein et al., 2011), the same mechanisms are presumed
to be important across species. Neurogenesis in the dentate gyrus, in particular, plays a role in
both the development of depression (Snyder et al., 2011) and antidepressant effect (Sahay and
Hen, 2007). Therapeutic interventions that induce a depressive phenotype, such as
corticosterone/glucocorticoid treatment (Brummelte and Galea, 2010), chronic stress (Pham et
al., 2003), interferon treatment (Kaneko et al., 2006) lead to a significant reduction in
neurogenesis. On the other hand, treatments that have an antidepressant effect increase
neurogenesis, whether it be pharmacologic, in the case of MAOi's (Malberg et al., 2000;
Nakagawa et al., 2002a), Tricyclics, SSRI's (Malberg et al., 2000; Santarelli et al., 2003), SNRI's
(Shankaran et al., 2006), Lithium (Chen et al., 2000), antipsychotics (Kippin et al., 2005), or
electroconvulsive treatment (Madsen et al., 2000).
Antidepressants' effects on already-born neurons
16
Antidepressants have also been shown to have effects on neurons independent of
neurogenesis. Newly born neurons have a higher rate of survival in the presence of
antidepressants, independent of the effect of SSRI's on progenitor proliferation (Bath et al.,
2012). SSRI administration results in structural changes in non-neurogenic brain regions
(McCabe and Mishor, 2011). IV Ketamine, currently under investigation in clinical trials for
antidepressant treatment, is also known to facilitate the massive reorganization of the dendritic
architecture of neurons from numerous cortical brain regions (Surget et al., 2011).
Schizophrenia: Antipsychotics
The treatments for bipolar disorder and schizophrenia fare better than placebo (Leucht
et al., 2012) but their efficacy is limited to only a subset of symptoms, and also come with a
high-side effect profile that often results in treatment discontinuation, either by the choice of the
patient or due to the development of dangerous side effects (Daumit et al., 2008; Caroff et al.,
2011).
The first antipsychotic, chlorpromazine, was discovered in 1952, rather accidentally: it
was initially developed for use as a surgical anesthetic, but was used in psychiatric patients for
its sedative effects. The discovery of chlorpromazine led to a revolution in psychiatric care, as
patients that responded to treatment were discharged from confinement in psychiatric inpatient
hospitals: previous to this, the diagnosis meant a lifetime of respite and confinement in these
wards. The current pharmacological treatments for schizophrenia, referred to as the
antipsychotics have some antagonist activity at the dopamine D2 receptor. The pharmacologic
agents available to date for the treatment of the disorder only provide symptomatic relief, and
are effective for only a portion of the symptoms (mainly positive). The treatments themselves
have significant adverse effects, including tardive dyskinesia, sedation, metabolic changes
including diabetes, and because of this, discontinuation rate is high (Salimi et al., 2009). The
17
current pharmacologic treatments for bipolar disorder include a combination of mood stabilizers
plus an antipsychotic (Correll et al., 2010).
Current hypothesis pertaining to risk for major depressive disorder (MDD)
There are many hypotheses as to what results in the manifestation of these
psychiatric conditions, based on neurochemical, neuroanatomical, and epidemiological findings,
the pharmacology or effect of treatments, and, most recently, genome-wide association studies
(GWAS). The leading hypothesis is that there isdysfunction of numerous brain areas related to
the generation and processing of emotions, such as the prefrontal cortex, amygdala,
hypothalamus, medial septal areas and the hippocampus. Significant efforts have been made to
examine the role of neurogenesis on the etiology of depression. This is based on the findings
that antidepressants, as well as other interventions that have antidepressant like effects such as
deep brain stimulation and environmental enrichment in animal models, all increase the number
of newly-born neurons in the dentate gyrus, and because the time course for the maturation of
adult-born neurons is consistent with the onset of antidepressant effectiveness. However, a
change in adult neurogenesis is not sufficient to explain all the activity of antidepressants, as
there are neurogenesis-independent actions of antidepressants (Sahay and Hen, 2007).
Furthermore, research to date suggests that adult neurogenesis in the hippocampus is a
substrate for the behavioral effects of antidepressants, but is not an essential contributor to the
etiology of depression. Two large-scale genome-wide association studies have been conducted
to date with no significant correlation identified between disease and a genomic region (Muglia
et al., 2010). Thus, the etiology of unipolar depression remains a significant challenge to
address.
Current hypothesis pertaining to risk for bipolar disorder
The concordance rate of bipolar I disorder in monozygotic twins is around 40%
compared to -10% in dizygotic twins and in non-twin siblings (Kieseppa et al., 2004). The
18
overall heritability of bipolar disorder is 0.71 (Edvardsen et al., 2008). There have been several
genome-wide association studies to date that have identified novel genetic loci associated with
the disease (2007; Ferreira et al., 2008; Sklar et al., 2008; Purcell et al., 2009). These studies
point to the heterogeneity of underlying genetic contributions to the risk of developing bipolar
disorder. Recent studies have identified linkage disequilibrium for polymorphisms within the
CACNA1C L-type voltage gated calcium channel subunit and ANK3 (Ferreira et al., 2008), as
well as other genetic regions close to miR137 (Ripke et al., 2011), CDH7 (Soronen et al., 2010),
and clOorf26 open reading frames (Ferreira et al., 2008; Sklar et al., 2008; Moskvina et al.,
2009). As ANK3 plays an important role in clustering Na+ and K+ channels at the axon initial
segment (Pan et al., 2006), it is tempting to speculate that changes in channel property or
synaptic transmission contribute to the etiology of bipolar disorder. There is, however, a
significant proportion of the heritability of the disorder that has yet to be taken into account,
illustrating the need for novel and, as yet unexplored mechanisms to probe for disease
heritability that go beyond the sequence of the genes themselves.
Current Hypotheses on Disease Etiology: schizophrenia
Numerous hypotheses to the etiology of schizophrenia have been postulated to
date such as the dopamine, glutamate, and GABA hypotheses, based on medications,
pharmacological mimicry and postmortem neuropathological findings, but none are sufficient to
explain the onset of the syndrome. The neurodevelopmental hypothesis for schizophrenia has
also been gaining traction in the field. This hypothesis predicts that alterations during the
development of the brain result in cognitive deficits and renders the individual more susceptible
to developing psychiatric disorders at a later age. Indeed, the cognitive deficits seen in patients
with schizophrenia are often detected long before the diagnosis is made, and these same
deficits are often seen in family members (Gold and Weinberger, 1995). Though there are no
pathomnemonic features or tests to date for the disorder, and there is no identified organic
19
cause, several lines of evidence suggest that such a cause may exist. Endophenotypes,
including those from neurological testing and functional brain imaging have been identified in
some people with schizophrenia and their family members. These endophenotypes may also
point to a brain with more susceptibility for developing schizophrenia. Genetic abnormalities that
result in aberration of neural development have been associated with schizophrenia, as will be
discussed below.
There is a 50% concordance rate for schizophrenia between monozygotic twins, and the
offspring of affected individuals have a ten-fold increase in risk for developing the disorder
relative to the general population, highlighting a genetic basis for the disorder. Recent genetic
evidence indicates that microdeletions or duplications of certain chromosomal loci, known as
copy number variations (CNVs), increase the risk for schizophrenia. Chromosome 22q1 1.2
microdeletions occur in 1 of every 4,000 live births. These deletions produce a wide range of
clinical presentations including velo-cardio-facial syndrome, DiGeorge syndrome, and mental
retardation (Debbane et al., 2005). About 30% of carriers develop psychosis (Debbane et al.,
2006a). Reduced brain volume, or microcephaly, is a common pathological feature of these
carriers (Debbane et al., 2006b). Recently, additional CNVs have been identified that increase
the risk for schizophrenia including microdeletions of chromosome 1q21.1, 15q13.3, 15q11.2,
3q29, 16p13.1, and 17p12 and microduplications at 16p11.2 (International Schizophrenia
Consortium2008; Stefansson et al., 2008a; Vacic et al., 2011). Interestingly, 1q21.1
microdeletions and duplications are also involved in other neurodevelopmental disorders such
as microcephaly/macrocephaly with behavioral abnormalities (Brunetti-Pierri et al., 2008) and
autism (Mefford et al., 2008), whereas 15q 13.3 microdeletions and duplications are associated
with idiopathic epilepsy, mental retardation, autism, ADHD, and anxiety disorder (Ben-Shachar
et al., 2009; Helbig et al., 2009). Another study identified rare CNVs disrupting multiple genes in
neurodevelopmental pathways in schizophrenia (Walsh et al., 2008). Collectively, genetic
20
evidence strongly supports the notion that impaired brain development increases the risk for the
manifestation of mental illnesses. The genetic findings are also consistent with the general
description of the neuropathology of schizophrenia, namely enlarged lateral ventricles and
reduced cortical/hippocampal size (Tamminga and Holcomb, 2005). It remains to be determined
why a given genetic lesion gives rise to clinically heterogeneous phenotypes.
A Common set of heritable risk for psychiatric disorders
There is now considerable evidence for a common set of heritable risk for psychiatric
disorders including schizophrenia, bipolar disorder, as well as autism. For starters, the
concordance between monozygotic twins, and the increased incidence amongst family
members, for bipolar disorder and schizophrenia support the existence of a such a common
genetic factor (Lichtenstein et al., 2009). There are a number of recurrent CNV's that are
common to both schizophrenic and autistic patients, albeit some go in opposite directions,
suggesting a possible gene-dosage effect (Mefford et al., 2008; Stefansson et al., 2008a;
Moreno-De-Luca et al., 2010; Malhotra and Sebat, 2012; Rodriguez-Murillo et al., 2012) Further
evidence for the shared genetic risk for these psychiatric disorders comes from genome-wide
association studies (GWAS) that have identified numerous genetic risk SNPs that are common
between disorders(Sklar et al., 2008; Ripke et al., 2011). Current evidence suggests that
schizophrenia, bipolar disorder, and autism, either treated separately or treated as a unitary set,
would have an inheritance pattern similar to that of several non-neuropsychiatric disorders,
including adult-onset diabetes and inflammatory bowel disease, all of which follow a complex
inheritance pattern. It is interesting that schizophrenia has common risk variations for both
autism and bipolar disorder, both in SNP alleles and recurrent CNV's, almost as if it is an
intermediary phenotype.
Putting everything together: biochemical pathways.
21
Many of these risk alleles affect genes implicated in neuronal development with known
roles at an early stage; these genes would have an effect on the individual before the onset of
overt symptoms or diagnosis. Furthermore, many of the genes identified through GWAS are
known to participate in established pathways that are relevant for neuronal development and
function.
One could hypothesize that the risk for developing psychiatric disorders can be
accounted for by disruptions to specific biochemical pathways. This type of approach has been
successfully implemented for cancer treatment, where the identification of disease targets
based on alterations in pathways has led to the development of targeted therapeutics (de Bono
and Ashworth, 2010). This would enable the classification of patients based on the presence of
risk, grouped by biochemical pathways, as well as provide a more tractable predictor of patient
response to medications. Some of the recently identified risk factors affect neural development
at an early stage; before the onset of overt symptoms leading to diagnosis with the disorder.
Importantly, many of the genes that have been identified through these genetic studies encode
proteins that are known to be pivotal for signaling pathways associated with neuronal
development and function. It is important then to address the causality between these signaling
pathways that are important for neurodevelopment, and the risk of developing neuropsychiatric
disorder.
Several pathways have been clearly implicated in neuronal development: these include
the Wnt, Akt, Lis1/NudeL, PKA, and EGR/Map Kinase pathways. These signaling pathways are
not straightforward; there is substantial parallel signaling, as well as feed-forward and feedback
signaling within one biochemical pathway, as well as substantial crosstalk between these
pathways.
22
In the following paragraphs I will briefly identify some pathways important for neuronal
development. I will elaborate on the Wnt pathway moreso than the others because of the
intriguing, and notably undocumented, number of genes that are in or near affected gene
regions that converge onto the pathway: it is not meant to diminish the importance of the other
pathways in neuronal development.
cAMP-PKA-CREB pathway
The cyclic adenosine monophosphate (cAMP)-Protein Kinase A (PKA)-cyclic-AMP
response element binding protein (CREB) pathway is a pathway that is activated upon the
binding of ligands to G-protein coupled receptors (GPCRs) that are coupled to the GasGpy
heterotrimeric G-protein complex. Inthe baseline state, the G-protein complex is bound to GDP
and is inactive. Upon ligand binding to these GPCR, there is an exchange of GDP to GTP,
leading to the dissociation and activation of Gas. Gas -GTP binds to stimulate the activity of
adenylyl cyclase, which converts adenosine triphosphate (ATP) into cyclic adenosine
monophosphate (cAMP). cAMP acts as a second messenger, and the increase in the levels of
cAMP lead to the activation of several key downstream events. cAMP can bind to and open
cyclic-nucleotide gated ion channels, influencing neuronal conductance. It can also activate
exchange proteins activated by cAMP (EPACs). It's most well characterized function is the
activation of protein kinase A (PKA). PKA normally exists as a holoenzyme consisting of two
regulatory and two catalytic subunits. cAMP binds to the regulatory subunits, releasing the
catalytic subunits for downstream activity. PKA phosphorylates a plethora of substrates: one of
them is CREB, a transcription factor that binds to the cAMP response element (CRE).
Phosphorylated CREB (pCREB) induces the transcription of many genes involved in neuronal
development. pCREB is found in almost all newborn neurons and correlates with the
expression of doublecortin (Nakagawa et al., 2002a; Nakagawa et al., 2002b), and is important
for newborn neuron survival(Herold et al., 2011). However, the role of CREB in the proliferation
23
of neural precursors is yet unknown (Merz et al., 2011). However, it is well established that
increasing cAMP levels by inhibiting the phosphodiesterase 4 family of enzymes with rolipram,
does increase the rate of neuronal progenitor proliferation, raising the possibility that there are
other factors that contribute downstream of PKA (Li et al., 2009).
The P13K-AKT-mTOR pathway
The phosphatidylinositol (3,4,5)-triphosphate kinase (Pl3K)-AKT/PKB-mammalian Target
of Rapamycin (mTOR) pathway is activated by another class of membrane-bound receptors, the
receptor tyrosine kinases. In the baseline state, AKT is bound to phosphatidylinositol(3,4)bisphosphate (PIP2). Upon activation of a receptor tyrosine kinase, or, in some cases, a Gprotein coupled receptor, P13K phosphorylates PIP2 to form PIP3. AKT bound to PIP3 is then
phosphorylated by mammalian target of rapamycin complex 2 (mTORC2), then phosphorylated
by phosphinositide dependent kinase 1 (PDPK1). The phosphorylation by both kinases
activates AKT to then phosphorylate its targets, including mTOR, implicated in autism, GSK3p,
involved in the Wnt signaling cascade, BAD, a pro-apoptotic mitochondrial protein, and IKB
kinase (IKK), which regulates NFKB mediated transcription.
The intersection of the AKT pathway with the pharmacology of psychiatric disorder
treatment is notable. Dopamine d2 receptors, which are targets of all antipsychotics to date,
have been shown to modulate AKT signaling by recruiting a signaling complex that results in the
inactivation of AKT. Upon D2 receptor activation and phosphorylation by G-protein coupled
receptor Kinase (GRK), P-arrestin and PP2A are recruited to the cell membrane, where they
interact with and dephosphorylate AKT. Antipsychotics inhibit this process, allowing AKT to
remain active(Beaulieu et al., 2005; Freyberg et al., 2010).
Lithium, a treatment for bipolar disorder, is thought to exert its action by acting to
activate AKT activity on GSK3P. Lithium has been proposed to solubilize AKT from the P24
arrestin/PP2A complex(Beaulieu et al., 2004). Thus, both these treatments secondarily affect
the wnt pathway via affecting AKT activity.
PLC-PKC pathway
Phospholipase C is an enzyme that is activated downstream of GaoGpywhich cleaves
phosphatidylinositol 4, 5 bisphosphate (PIP2) into Inositol-3-phosphate (IP3) and diacylglycerol
(DAG). IP3 binds to receptors that regulate the release of calcium from intracellular stores, such
as the smooth endoplasmic reticulum, leading to elevated levels of intracellular calcium, and
subsequently activating calcium-dependent signaling cascades such as those involving
calcium/calmodulin-dependent protein kinase i (CaMKII), calcineurin and PKC.
Map Kinase/ ERK pathway
The mitogen associated protein kinase (MAPK)/ ERK pathway refers to a general
pathway in which a receptor-linked tyrosine kinase activation by ligand activates the GTPase
Ras. Ras then activates a MAP kinase kinase kinase (MAP3K), which activates, MAP kinase
kinase, which activates MAP kinase (MAPK). MAPK's activate transcriptional activity of prosurvival and proliferative genes via several mechanisms. One mechanism is the
phosphorylation and activation of RSK, a40s ribosomal protein S6 kinase. This results in the
alteration of RNA translation. RSK also phosphorylates CREB as well as c-Myc, c-Fos, NfKB,
altering transcription of survival genes. Notably, RSK also phosphorylates GSK3P.
ERK2 is known to be essential for neuronal progenitor proliferation. ERK2 (MAPK1)
knockouts(Satoh et al., 2011) die during embryogenesis. Conditional knockout of ERK2 in
neuronal progenitors results in mice with decreased cortical thickness, and have a pool of
undifferentiated neuronal progenitors (Samuels et al., 2008).
The Wnt signaling pathway
25
Wnts are secreted lipoproteins that act as morphogens and play important roles in the
development and patterning of various limbs and organs. The Wnt ligands bind to their cell
surface receptor, the Frizzled (FZ) family of seven transmembrane G-protein coupled receptors.
The Wnt ligands were identified by their homology to Wnt, which was named from a
combination of the drosophila gene wingless (Wg) and its mammalian homolog Integration 1
(Int1). To date, nineteen WNTs and ten FZD family members have been identified in humans.
The various Wnts as well as the FZDs have distinct, yet overlapping expression patterns
throughout the body, which is thought to account for the variability of phenotypes that result
upon loss of function (http://www.tcd.ie/Zooloqy/research/WntPathway/wnt.php, accessed April
17, 2012). Furthermore, it has also been shown that different Wnts have different affinities for
the different FZs, and that different Wnts activate different downstream signaling cascades
(Kikuchi et al., 2009).
Though none of the Wnts have been crystallized for structural analysis, much is known
about their general structure. The Wnts are all about 320-400 amino acids long. They all have
an N-terminal signaling sequence, which is required for their secretion, followed by a conserved
domain consisting of 22-24 cysteines, the first of which is palmitoylated. The secreted form of
the protein is highly hydrophobic (Mikels and Nusse, 2006). The loss of palmitoylation reduces
Wnt 's potency, which can be overcome with high concentrations of Wnt, suggesting that
palmitoylation is required for Wnt localization(Nusse, 2003; Mikels and Nusse, 2006). Numerous
glycosylated forms of Wnt proteins have been detected, supporting the posttranslational
modification of Wnts as a means to regulate Wnt signaling.
The ten members of the frizzled family of seven transmembrane G-protein coupled
receptors share an N-terminal extracellular domain, three extracellular and three intracellular
loops, and an intracellular C-terminal tail, like the classical G-protein coupled receptors;
however many features make the FZD family unique. The N-terminus contains a domain rich in
26
cysteines, and Wnts bind to this region (Schulte and Bryja, 2007). The extracellular loops have
putative glycosylation sites, a posttranslational modification that is known to play a role in both
receptor localization and ligand binding. The intracellular C-terminal region of the receptor
contains a PSD-95/DISC Large/POZ-1-homologous (PDZ) binding domain that is crucial for
protein-protein interactions needed to mediate its downstream effects. Furthermore, the
intracellular loops have numerous consensus sites for serine/threonine as well as tyrosine
phosphorylation, reminiscent of receptor tyrosine kinases. There is evidence to suggest that
FZD receptors exist as dimers which is facilitated by the cysteine-rich domain, and Wnt ligand
binding. Moreover, FZD dimerization appears to be sufficient for activation of downstream
pathways (Carron et al., 2003).
Extensive work in the past 20 years has led to the identification of several Wnt signaling
pathways downstream of Wnt ligand binding to FZD or one of its co-receptors: lipoprotein related
protein 5/6 (LRP5/6) (Pinson et al., 2000; Tamai et al., 2000; Wehrli et al., 2000), receptortyroine kinase-like orphan receptor 2 (Ror2) (Saldanha et al., 1998), and related to receptor
tyrosine kinase (Ryk) (Inoue et al., 2004).
Canonical Wnt signaling
Binding of Wnt to LRP5/6- associated frizzled activates a pathway known as the
canonical Wnt signaling pathway. The canonical Wnt signaling pathway is defined by their
dependence on the transcription co-activator p-catenin and the downstream signaling cascades
that are associated with it. Briefly, Fzd proteins have a conserved KTxxxW domain that is
required for interaction with dishevelled (DVL) proteins. In the absence of Wnt stimulation, DVL
is part of a destruction complex, in conjunction with AXIN, adenomatous polyposis coli (APC),
casein kinase 1 alpha (CK1a), and glycogen synthase kinase 3 (GSK3p). This destruction
complex binds and sequentially phosphorylates p-catenin at multiple sites, thus increasing the
27
affinity of P-catenin to P-TrCP. p-TrCP serves as one of the F-box proteins in the Skpl-CullinlF-box-protein (SCF) E3 ubiquitin ligase complex. Binding of p-TrCP to p-catenin leads to the
polyubiquitination and subsequent degradation of P-catenin, preventing the accumulation of pcatenin and limiting its downstream function. Upon Wnt stimulation of the FZD-LRP5/6 complex,
DVL is recruited to the complex, where it polymerizes, and through its DIX domain binds to the
DIX domain of AXIN. This, in turn, leads to the recruitment of AXIN towards the FZD-LRP5/6
complex, where it mediates interaction with of CK1 a and GSK3P to LRP5/6. Next, the kinases
phosphorylate LRP5/6 and increases its affinity for AXIN. The LRP5/6 interaction directly inhibits
GSK3p phosphorylation of P -catenin (Piao et al., 2008), one of the priming phosphorylations of
p-catenin, disrupting the ability of the destruction complex to target p-catenin for ubiquitination
and subsequent degradation. Thus, the activation of the Wnt pathway leads to an increase in
cytosolic P-catenin.
p-catenin is a transcriptional co-activator of TCF/LEF transcription factors, which control
the dynamics of the cell cycle via regulation of cyclinD1. The accumulation of p-catenin in the
cytosol leads to its increase in the nucleus and the binding of P-catenin and it's associated
transcriptional activators to the T-cell factor and lymphoid enhancer-binding protein (TCF/LEF)
DNA binding protein family. TCF/LEF has three identified domains, a high mobility group (HMG)
box domain that is crucial for DNA interaction, a caspase activated deoxyribonuclease (CAD)
domain necessary for binding to Groucho/TLE, and an N-terminal P-catenin binding domain. In
the absence of nuclear P-catenin, the TCF/LEF occupying TCF/LEF DNA binding sites is bound
to tetrameric Groucho/TLE, which recruits transcriptional repressors, such as histone
deacetylases (HDACs), keeping the DNA in a condensed, repressed state. P-catenin directly
competes with, and displaces Groucho/TLE (Daniels and Weis, 2005), and recruits general
transcriptional activators such as the histone acetyltransferase CBP, the SWI/SNF complex
protein Brg-1, and TATA- binding protein, as well as a more specific core complex consisting of
28
Pygopus and Legless/BCL9 (Belenkaya et al., 2002; Kramps et al., 2002; Thompson et al.,
2002). CBP acetylates histone residues in the promoter region, and the recruitment of Pygopus
and Legless/BCL9 leads to the methylation of H3K9 residues by SET-1 methyltransferases,
altering the chromatin structure from a condensed, transcriptionally repressed one, to a more
relaxed and open state, thus allowing transcription (Fiedler et al., 2008).
The canonical Wnt pathway and psychiatric disease gene candidates
Several players in the Wnt signaling pathways have been associated with psychiatric
disease. CHD8, which is also recruited during this process, acts to counter the activating
activity of p-catenin, by binding to p-catenin and also Histonel, and thus limiting the relaxation
of chromatin. CHD8 SNPs are associated with autism, and BCL9 is one of the genes in the
1q21.1 recurrent CNV, with microdeletions associated with schizophrenia and microcephaly and
microduplications associated with autism and macrocephaly. Furthermore, ADA2A, in the 17q12
microdeletion associated with autism, is known to be an acetyltransferase required for
transcriptional activity and proliferative effects mediated by p-catenin (Yang et al., 2008).
Both the wnt ligands and the receptor are glycosylated, and perturbations of
glycosylation are known to affect the function, localization and signaling through them. SNPs in
genes that are involved in glycan synthesis (hsa0l 030) associate with schizophrenia. Ankyrin 3,
which has noncoding SNPs within its gene that associate with both schizophrenia and bipolar
disorder, is known to be a component of the Cadherin complex, and disruption of the cadherin
complex is known to lead to mislocalization of p-catenin (Orsulic et al., 1999).
DLG1 (Sap97) is one of the interactors of Fz and is known to signal downstream of wnt
binding by binding to Adenomatous Polyposis Coli (APC), affecting cell proliferation: this gene is
found in the 3q29 CNV deletion. ErBB4/Neuregulin, another well-studied candidate that
involves a secreted protein ligand and surface receptor, is a receptor tyrosine kinase, and its
29
family members have been shown to activate cascades that phosphorylate and lead to the
intracellular localization of p-catenin: overexpression of ErbB4 Cyt2 incresed nuclear p-catenin
and TCF-LEF transcriptional activity (Muraoka-Cook et al., 2009). PAK2, also in the 3q29 CNV,
also serves to phosphorylate and promote the nuclear localization of p-catenin (Zhou et al.,
2011).
In addition to the recruitment of DVL, Wnt activates the G-proteins associated with Fz
proteins. The FZDs are coupled to the Gao GPy trimeric G-protein complex (Katanaev et al.,
2005), and Wnt binding to Fz liberates the Gpy subunits to activate a calcium-dependent
pathway that involves the activation of phospholipase C cascade. The perturbation of these
signaling pathways can have consequences beyond this pathway, and can impact the canonical
Wnt pathway as well. Neurogranin, implicated in bipolar disorder and schizophrenia, is a key
regulator of calmodulin, limiting its activity by binding to it; its binding to calmodulin is dependent
on its PKC phosphorylation. The association of voltage-gated calcium channel, CACNA1C,with
bipolar and schizophrenia is another intriguing risk gene related to calcium signaling.
Binding of Wnt to Fz leads to activation of pathways involved in planar cell polarity, acting
through dishevelled, activating RhoA and RACI (Gao et al., 2011; Shafer et al., 2011) (Bo et al
2011 Developmental Cell). RAC1 activation then activates c-Jun N-terminal kinase (JNK)
downstream of Wnt5a (Oishi et al., 2003). JNK activation has been shown to be crucial for the
proper development of axons and dendrites (Rosso et al., 2005)Calderon et al 2012), and to
enhance or inhibit canonical Wnt activation, depending on the Wnt ligand (Billiard et al., 2005).
Tao Kinase 2, in the 16p1 1.2 region, which has CNV's that associate with both autism and
schizophrenia (deletion and duplication, respectively), also plays a role in the activation of JNK.
One of the alternative, p-catenin independent pathways that is downstream of LRP5/6Frizzled signaling, depends on the inhibition of GSK3P. GSK3P is known to destabilize
30
microtubules by phosphorylating microtubule proteins such as TAU, MAP1 B, and MAP2.
Inhibition of GSK3p stabilizes microtubules, promoting axon growth and growth cone
remodeling. In this manner, the wnt pathway is able to exert effects on neuronal morphology.
Binding of Wnt to Ryk has been shown to directly activate the Src pathway, similar to the
activation of many other receptor tyrosine kinases such as EGR. The activation of Src, in turn,
has been shown to be crucial for axon guidance. Ryk has also been shown to interact directly to
FZD8 XXXXXX), as well as DVL, and to potentiate the canonical Wnt signaling pathway,
suggesting that the presence of Ryk in conjunction with Frizzled may either stabilize the DVL-Fz
interaction, or enhance AXIN interaction with Fz. The convergence of neuropharmacological
and genetic evidence makes the wnt pathway(s), if not a promising target, at least a convenient
framework within which the function of psychiatric risk gene polymorphisms and rare variants
can be tested.
31
Nrg1
P-cat
Src
-cat
Fer
t
cat
9O
cytoplasm
Nucleus
Proteasomal
degradation
Figure 1. Canonical WNT pathway in inactive state. Tyrosine phosphorylation of p-catenin
results in dissociation of p-catenin from its membrane interactors and results in cytosolic
localization of p-catenin. And binding to destruction complex. Sequential serine phosphorylation
by CK1 and GSK3p (represented as red P's). p-catenin is subsequently polyubiquitinated and
degraded by the proteasome.
32
Antipsychotics
Gc
DA
DRQ2
L/L,,
K.]'
4
~cat
0-at
-cat
-Mat
Cytoplasm
SNucleus
0-ca
ADA
E-Cadherin
Cyclin D1
Tbx1
Figure 2. Activated canonical Wnt pathway, and select interactions of pathway with
psychiatric disorder therapeutics and risk genes. Wnt-Fz/LRP5/6 interaction recruits
destruction complex to the cell surface, and CK1/GSK3p is inactivated. Cytosolic p-catenin bind
does not get degraded, gets phosphorylated by PKA, translocates to nucleus where it binds to
TCF/LEF transcription factors, recruiting BCL9, Pygo, CHD8, ADA2A, CBP to initiate polil
mediated transcription. Lithium, antipsychotics increase AKT activity, which increases its
inhibition of GSK3p, enhancing the activity through this pathway. Phosphodiesterase inhibitors
would increase PKA activity, which enhances this pathway by increasing nuclear p-catenin
translocation and by inactivatinq GSK313.
33
Disrupted in Schizophrenia 1 (DISC1): a single gene to which many pathways converge
Disrupted in Schizophrenia-1 (DISC1) was identified as the gene that was disrupted on
chromosome 1 in a Scottish family with a high concordance of major psychiatric disorders and a
balanced translocation between chromosomes 1 and 11 (Millar et al., 2000). Karyotyping on five
generations of this family revealed 18 of the 29 members with this translocation have
schizophrenia, recurrent major depression, or bipolar disorder. This type of near Mendelian
segregation for psychiatric disorders had not been previously observed. Mouse models for
DISC1 have been generated, and the mice display a variety of phenotypes that can be
considered schizophrenia-like behavioral deficits. Mouse models expressing a transgene of
human DISC1 mimicking the Scottish translocation mutant exhibit increased ventricle size,
decreased gray matter volume, and changes in dendritic arborization in cortical and
hippocampal neurons (Hikida et al., 2007; Li et al., 2007; Pletnikov et al., 2008; Shen et al.,
2008). These mice also exhibit behavioral abnormalities such as hyperactivity (Hikida et al.,
2007; Pletnikov et al., 2008), increased immobility in the forced swim test (Hikida et al., 2007),
decreased sociability (Pletnikov et al., 2008), and working memory (Kvajo et al., 2008; Pletnikov
et al., 2008). Clapcote et al characterized ENU induced mouse DISC1 exon 2 mutants and
reported that the Q31L mutant exhibits depression-like and the L100P mutant exhibits
schizophrenia like behavior (Clapcote et al., 2007). In addition, reduced embryonic and adult
hippocampal neurogenesis have been reported in a DISC1 BAC transgenic mouse model and
DISC1 exon 7-8 deletion mutant mice (Kvajo et al., 2008; Shen et al., 2008). Finally, DISC1 is
implicated in the proper integration of adult born neurons in the dentate gyrus circuit (Duan et al.,
2007). Collectively, the neuropathology and behavioral phenotypes exhibited by the various
DISC1 mouse models strongly supports a role for DISC1 in mental health.
DISC1 binding partners
34
Since its identification as a psychiatric disease risk gene, a large number of DISC1
binding partners have been identified. Some validated interacting partners for DISCI include
NDE1/NDEL1, important for neuronal migration, Kalirin-7, important for glutamatergic signaling,
ATF4/5, important for neuronal progenitor proliferation, the PDE4family of phosphodiesterases,
implicated in depression, and GSK3p, a target of Lithium used in bipolar disorder treatment.
These interactions will be described in more detail below.
DISC-1 Structure, features, and Interactions
1:11 Translocation Breakpoint
T1 Transcript
854
sRNA1
shRNA2
4441bp5'TR
Protein structure
A
331-34
166-394
L
ft.DISC-1
&SS-"
W02-830
607-628
Interaction Partners
(Binding regions)
*fl
31-65
101-135 190-230
PfDE 46
4
266-290
JE02.
902-915
459-479
K<
Leucine-Zipper
Coiled-Coil
Phosphorylation site
Nuclear Localization Sequence
Figure 3. Schematic of validated DISCI partners. The interactions relevant for further
chapters are colored.
35
DISC1 and neuronal migration
DISC1 binds to nudE nuclear distribution gene E homolog-like 1 or NDE1/NDEL1, which
is known to play a role in neuronal migration, proper neuronal localization and neurite and
axonal development (Kamiya et al., 2005). Disrupting DISC1/Ndel1 interaction disrupts neurite
outgrowth in PC12 cells (Pletnikov et al., 2007), and also prevents the colocalization of DISC1
and Lis1, which localizes with the dynein heavy chain, indicating that DISC1 plays an important
role in the function of Ndell/Lisl complex by regulating its localization. Our lab has shown that
DISCI also interacts with this complex via its interaction with DIX domain containing-1 (Dixdcl),
the third mammalian gene discovered to contain a Disheveled-Axin (DIX) domain (Dixdcl).
DISC1, Dixdcl and Ndel1 form a tripartite interaction, and the presence of either DISCI or
DIXDC1 seems to be able to properly localize Ndel1, indicating that these two proteins may
have overlapping/ redundant effects on neuronal migration (Singh et al., 2010). This also
indicates that a partial loss of function of either of these two genes can be offset by the function
of the other, potentially explaining why differences in the ability of DISC1 snps to bind to NDEL
does not result in an appreciable difference in human disease burden.
DISC1 and the WNT signaling pathway
DISC1 has also been shown to bind to, and inhibit the activity of GSK3-p, one of two
forms of an enzyme that is known to lead to the phosphorylation of many downstream proteins
that are crucial for the pathophysiology of many nervous system disorders. One GSK3-p target
is TAU, which forms aggregates in many nervous system disorders including Alzheimer's (TAU
forms the neurofibrillary tangles that are used in the Braak and Braak postmortem staging of
Alzheimer's), Parkinson's, and tuberous sclerosis: nervous system disorders in which there is
TAU aggregation are collectively referred to as tauopathies. Another GSK3-p target is P-catenin.
P-catenin is a crucial component of the canonical WNT signaling pathway. p-Catenin is bound
36
to a-catenin and the cadherin proteins at the cell membrane, as part of the adherens junction
(AJ) complex. p-catenin that is not bound to cadherins at the membrane binds to axin, which,
in conjunction with APC, complexes p-catenin with GSK3p. The formation of this complex leads
to the phosphorylation of p-catenin, which promotes its poly-ubiquitination and subsequent
proteasome-mediated degradation. When WNT binds to its receptor, frizzled, disheveled is
recruited to the membrane and activated. Activated disheveled inhibits GSK3-P, which
subsequently inhibits the phosphorylation and degradation of p-catenin, leading to its
accumulation in the cytosol and the activation of downstream pathways, such as the
transcriptions of genes which bind TCF/LEF transcription factors. The activation of this pathway
has been shown to be crucial for neuronal progenitor proliferation and the inhibition of the
premature differentiation of these neuronal progenitors, and deficits in this pathway have been
shown to result in microcephaly(Salcedo-Tello et al., 2011). By inhibiting GSK3p, DISC1
promotes neuronal progenitor proliferation, and the loss of DISC1 leads to decreased neuronal
progenitor proliferation (Mao et al., 2009; Ming and Song, 2009).
A molecular switch between the wnt signaling and neuronal migration function of DISC1
has been recently uncovered. DISC1 was found to be a target of PKA-mediated
phosphorylation, downstream of cAMP activation. The study identified two putative PKA
mediated phosphorylation sites on DISC1, and discovered that one of the sites, at serine 710, is
responsible for the differential localization and binding properties observed in neuronal
progenitors versus migrating neurons. DISC1 in neuronal progenitors is unphosphorylated at
this site, and binds to and inhibits GSK3p activity: upon phosphorylation at this site, DISC1
increases its affinity for BBS1 and localizes at the centrosome. Crucially, the authors
demonstrated that the phosphor-dead form of DISC 1 (S710A) rescued the proliferative but not
the migrational phenotype observed in DISC1 knockdown conditions, while the phosphor-
37
mimetic form of DISC1 (S710E) rescued the migrational aspects of DISC1 function(Ishizuka et
al., 2011).
DISCI and Kalirin-7: regulation of glutamatergic synapses
DISC1 plays a role in the regulation of dendritic morphology via its interaction with
Kalirin-7 a GDP/GTP exchange factor for Rac1. The DISC1/Kalirin-7 interaction is dependent
on NMDA-receptor mediated signaling, and acts to inhibit Kalirin-7 interaction with Rac1. The
loss of DISC1 function results in a short-term increase in dendritic spine size, and long-term
shrinkage of dendrites and a concurrent loss of surface AMPA receptors, accompanied by a
reduced frequency of miniature EPSC's (Hayashi-Takagi et al., 2010).
DISCI and ATF4
DISC1 also binds to Activating transcription factors 4 and 5 (ATF4 and ATF5,
respectively), basic-region-leucine zipper domain containing transcription factors of the
CREB/ATF family (Morris et al., 2003). ATF4, also known as CREB2 is both a transcriptional
activator and repressor that binds to CRE elements throughout the genome and is known to
play a role in hematopoiesis, osteoblast differentiation, neuronal progenitor proliferation,
learning and memory, and behavior (Ameri and Harris, 2008; Frank et al., 2010). Disrupting
ATF4 interaction results in an alteration of CRE mediated gene transcription (Sawamura et al.,
2008), which is known to have a critical role in memory formation and affective behavior
(Bartsch et al., 1995; Chen et al., 2003; Green et al., 2008).
Previous work examining the interaction of ATF4 and DISCI was conducted using
overexpression studies in cell lines, and in the nervous system of drosophila. Sawamura and
colleagues found that DISC1 overexpression resulted in a decrease in transcription as assayed
using a luciferase assay that implemented the consensus CRE sequence as the promotor to
drive luciferase expression, and examining the effect of DISC1 overexpression on the activity of
38
this promotor (Sawamura et al., 2008). However, the endogenous function of the DISC1-ATF4
interaction remains unanswered, as knockdown of DISC1 was not assayed in this study. The
effect of endogenous DISC1 on behavior was likewise not addressed in this past study, as
drosophila has no endogenous DISC1 homologue (Sawamura et al., 2008). A more recent
study has also undertaken the study of DISCI on ATF4 mediated transcription (Malavasi et al.,
2012), yet the study has still relied on overexpression assays, leaving the question of
endogenous DISC1 function unaddressed.
DISCI and the PDE4 phosphodiesterases
DISC1 has also been shown to bind to, and inhibit the activity of several isoforms of a
rolipram-sensitive, cAMP specific phosphodiesterase, grouped together as the
phosphodiesterase 4 (PDE4) family of genes. DISC1 has been shown to directly bind to, and
inhibit, at least some variant(s) of each of the four, A, B, C and D, PDE4 isoforms (Millar et al.,
2005; Cheung et al., 2007; Millar et al., 2007; Murdoch et al., 2007). The PDE4's, in particular
PDE4D, will be discussed in greater detail below.
PDE4D
The phosphodiesterase 4D gene is one of 4 genes that make up the PDE4 family of
phosphodiesterases (table X). The different genes that comprise the PDE4 family encode
distinct isoforms of a cyclic adenosine monophosphate (cAMP) specific and rolipram sensitive
phosphodiesterase. The gene locus that encodes each isoform contain multiple exons that span
dozens of kilobases of the genome, and each isoform has many distinct transcripts, known as
variants. All variants from an isoform contain a unique 5' exon which is believed to confer
distinct subcellular localization to the variants, and a common catalytic domain, which, as the
name suggests, converts cyclic AMP into non-cyclized AMP through a catalytic reaction (Ref).
39
The regulation of distinct variants within this gene is a subject of intense study ((DIaboga et al.,
2006; Li et al., 2009; De Arcangelis et al., 2010; Li et al., 2011b)refs).
In addition to the distinct functions conferred by the unique 5' exons, the
phosphodiesterase activity of this family of enzymes is carefully regulated through numerous
mechanisms intrinsic to the protein itself. The catalytic domain itself has a serine site that, when
phosphorylated by ERK/MAP kinase, inhibits the catalytic activity. The short variants
(PDE4D1/2) have the catalytic domain immediately following their unique exons, but the long
variants have an additional common regulatory domain that binds to and inhibits the activation
of phosphodiesterase activity by the catalytic domain (Houslay et al., 2005; Houslay, 2010)A
serine residue within the regulatory domain is phosphorylated by PKA, and this phosphorylation
triggers a release of the catalytic domain from binding to the regulatory domain(Zhang et al.,
2006a). This makes the long-isoforms of PDE4D's a PKA-dependent cAMP specific
phosphodiesterase.
DISC1 interacts with the cyclic AMP specific, rolipram sensitive phosphodiesterase 4
(PDE4) family isoforms through multiple interaction sites. Rolipram is effective as an
antidepressant in mice, but is not used in the United States for this purpose because of its side
effect profile, which includes nausea. Mice lacking PDE4B and 4D display a phenotype
reminiscent to mice on antidepressants (Zhang et al., 2002; Zhang et al., 2008). Previous work
has shown that DISC1 has multiple interaction domains with phosphodiesterase 4, and
interactions have been reported with isoforms A, B, C and D (Millar et al., 2005; Murdoch et al.,
2007). Elevated levels of cAMP activity is able to disrupt the interaction of full length DISC1 with
isoform C variant 2 and isoform Dvariant 3, but does not affect binding to isoform A variant 5
and isoform B variant 1. It is presumed that the binding characteristics are consistent within the
different variants within the isoforms described. A peptide array binding characterization utilizing
various fragments of DISC1 and PDE4 B and Dhave shown that compared to PDE4D, PDE4B
40
has multiple unique binding sites and it is believed that these additional binding sites confer
stability to PDE4A and B compared to C and D. In contrast to full-length DISCI, a 71kb form of
DISC1 dissociates from PDE4B under cAMP/PKA stimulation, and the dissociation of PDE4B
from DISC1 results in an increase in its phosphodiesterase activity.
41
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54
CHAPTER 2:
DISC1 acts as a co-repressor to inhibit ATF4 mediated transcription at the PDE4D
loci
Takahiro Soda, Christopher Frank, Alyssa Baccarella, Yu Park, Zack Flood, Cillian King,
Sangki Park and Li-Huei Tsai.
55
ABSTRACT
Disrupted in schizophrenia-1 (DISC1) is the gene on chromosome 1 that is disrupted
due to a balanced translocation between chromosomes 1 and 11. The LOD score for having this
translocation and having schizophrenia, bipolar disorder, or major depressive disorder is 7.1.
DISC1 has been shown to interact with numerous protein partners and to affect multiple
biochemical signaling pathways that impact neuronal development and function. One such
protein is activated transcription factor 4 (ATF4), also known as cyclic adenosine
monophosphate response element binding 2 (CREB2), a transcription partner that is crucial for
the maintenance of the neuronal progenitor pool. DISC1 has been shown to inhibit ATF4
mediated transcription from CRE binding sites, as assayed by artificial luciferase expression
assays; however the role of DISC1 on endogenous ATF4 function remains unclear. In this study
we identify a novel ATF4 and DISC1 binding region within the phosphodiesterase 4D gene
(PDE4D). PDE4D is itself a gene implicated in psychiatric disorders. Here, we demonstrate that
DISC1 interacts with this region to repress the expression of a unique variant of the gene,
PDE4D9. We further show that PDE4D9 expression is also regulated via Gs G-protein coupled
receptor signaling, and that this regulation is mediated by a novel mechanism in which DISC1
dissociates from this locus upon Gs stimulation. Our results suggest that a fraction of DISC-1
dysfunction can be accounted by the increase in PDE4D9 and that PDE4D9 may serve as a
target for novel therapeutic approaches.
INTRODUCTION
Disrupted in schizophrenia-1 (DISC1) was identified in 2000 (Millar et al., 2000) as a
gene product that becomes disrupted on chromosome 1 due to a balanced translocation
between chromosomes 1 and 11. Seventy percent of the family members that inherit this
translocation have major depression, bipolar disorder, or schizophrenia, with a combined LOD
score of 7.1 (Porteous et al., 2011). This type of Mendelian inheritance pattern had not been
56
previously observed in psychiatric disorders, and set off a flurry of investigations into the
function of this gene. The identification of numerous DISC1 binding partners (Millar et al., 2003;
Brandon, 2007; Camargo et al., 2007) has uncovered not only the function of DISC1 in several
signaling pathways, but has also implicated these pathways in psychiatric disorders. DISC1 has
served as a crucial pathfinder for psychiatric disorder risk genes; much as the genes identified
in rare neurodevelopmental disorders have served for the understanding of the molecular
players important for neuronal development. This has helped to set up a framework for
researchers to identify how newly identified risk alleles may contribute to disease and the
manifestation of disorder phenotypes.
Transgenic mice expressing the human DISC1 Scottish translocation mutation exhibit
increased ventricle size, decreased gray matter volume, and changes in dendritic arborization in
cortical and hippocampal neurons (Hikida et al., 2007; Li et al., 2007; Pletnikov et al., 2008;
Shen et al., 2008). These mice also exhibit behavioral abnormalities such as hyperactivity
(Hikida et al., 2007; Pletnikov et al., 2008), increased immobility in the forced swim test (Hikida
et al., 2007), decreased sociability (Pletnikov et al., 2008), and working memory (Kvajo et al.,
2008; Pletnikov et al., 2008). Clapcote and colleagues characterized ENU induced mouse
DISC1 exon 2 mutants and reported that the Q31 L mutant exhibits depression-like and the
L1OP mutant exhibits schizophrenia like behavior (Clapcote et al., 2007). In addition, reduced
embryonic and adult hippocampal neurogenesis have been reported in a DISC1 BAC transgenic
mouse model and DISC1 exon 7-8 deletion mutant mice (Kvajo et al., 2008; Shen et al., 2008).
Studies using acute knockdown of DISC1 products in mice have identified the role of DISC1 in
neuronal progenitor proliferation (Mao et al., 2009; Ishizuka et al., 2011), the proper integration
of adult born neurons in the dentate gyrus circuit (Kamiya et al., 2005; Duan et al., 2007;
Ishizuka et al., 2011), and neuronal migration (Ishizuka et al., 2011). Collectively, the
57
neuropathology and behavioral phenotypes exhibited by the various DISC1 mouse models
strongly supports a role for DISC1 in mental health.
Since its identification as a psychiatric disease risk gene, a large number of DISC1
binding partners have been identified. DISC1 interaction with GSK3p has been shown to be
crucial for its effects on neuronal progenitor proliferation, secondary to its effect of Wnt signaling
(Mao et al., 2009). DISC1 binds to centrosomal proteins, such as NDE1/NDEL1 (Brandon et al.,
2004; Bradshaw et al., 2008), Lis1, BBS1 and 4, and these interactions are important for proper
neuronal migration (Duan et al., 2007; Kamiya et al., 2008). The interaction between DISC1 and
Girdin/KIAA1212, an actin interaction protein, has been shown to be crucial for axonal
development, through the regulation of the AKT signaling pathway (Enomoto et al., 2005;
Enomoto et al., 2009; Kim et al., 2009). DISC1 modulates Rac1 GTPase activity by binding to
Kalirin-7, anchoring it to promote proper glutamatergic signaling (Hayashi-Takagi et al., 2010).
DISC1 has also been shown to regulate the activity of several isoforms of a cAMP specific,
rolipram-sensitive family of phosphodiesterases, referred to as the PDE4s (Millar et al., 2005;
Millar et al., 2007; Murdoch et al., 2007; Bradshaw et al., 2011; Carlyle et al., 2011). In summary,
DISC1 seems to be required for the proper localization and activity of its binding partners, and
the loss of DISC1 liberates its binding partners and allows for their unhindered activity.
DISC1 has been shown to associate with activating transcription factors 4 and 5 (ATF4
and ATF5), basic-region-leucine zipper domain containing transcription factors of the
CREB/ATF family of proteins (Morris et al., 2003). ATF4, also known as CREB2, is both a
transcriptional activator and repressor that binds to CRE elements throughout the genome and
is involved in diverse processes, including hematopoiesis, osteoblast differentiation, neuronal
progenitor proliferation, learning and memory, and behavior (Ameri and Harris, 2008).
Overexpression of DISC1 has been shown to decrease CRE-mediated activity, while loss of
DISC1 has been shown increase in the transcriptional activity of ATF4 (Malavasi et al., 2012).
58
However, we know very little about the effect of DISC1 dysfunction on endogenous targets of
ATF-4 mediated transcription. In this study, we identified a novel endogenous target of DISC-1
mediated transcriptional repression of ATF-4. This target region lies within the PDE4D gene
locus, and importantly, the effect of DISC1 mediated transcriptional repression is exquisitely
specific to a unique variant of PDE4D. Furthermore we identified a novel physiological
mechanism by which DISC-1 mediated transcriptional repression is regulated. Our results
suggest that a fraction of DISC-1 dysfunction can be accounted by the increase in PDE4D9 and
that PDE4D9 may serve as a target for novel therapeutic approaches.
RESULTS
Identification of ATF4-regulated Gene regions
DISC1 has been shown to lead to changes in gene transcription by modulating CRE
mediated transcription (Sawamura et al., 2008). Since DISC1 has no known DNA binding
domains, we hypothesized that DISC1 does this by binding to ATF4. To identify the targets of
DISC1 mediated transcription we conducted an unbiased chromatin-immunoprecipitation based
cloning strategy using a polyclonal ATF4 antibody developed in our lab (Frank et al., 2010)
(Figure 1A). Using this strategy, 100 loci were detected, and transcripts in the vicinity were then
identified by using the UCSC genome browser Blat. 12% of the binding loci lie within 1kb and 5'
of a nearest transcription start site, 10% within 1kb and 3' of the last known encoded exon, 46%
are located within introns, and 33 % are positioned more than 1kb away from any currently
identified transcripts, comparable to results obtained by studies using this method (Chen et al.,
2010) (Figure 1B). The identified regions were analyzed using Matinspector to search for
potential ATF4 binding sites. We subsequently confirmed three ATF4 binding regions through
ChIP-PCR from wildtype mice, PDE4D, TrpC, Sema4a, (Figure 1C). Importantly, these regions
could not be detected using ChIP-PCR on samples from ATF4 knockout mice (data not shown).
59
A.
Neuronal nuclear ysis
Fragmentaton of genome by
sonication
Protein digestion
Ptnenovnoroform extraction,
ethanol precipitation of DNA
T7 based sequencing of regions
B.
C.
ATF4
PDE4D
M
IgG
Input
gbow
TrpC
Sema4a
Figure 1: Identification of PDE4D loci as an ATF4 binding site. A. Schematic summarizing
our cloning strategy. B. Summary of the distribution of ATF4 binding sites relative to nearest
known coding region, identified by Blat search on the UCSC genome browser, mouse genome
mm9, based on NCBI build 37. C. Confirmation of several ATF4 binding regions. PDE4DPhosphodiesterase 4D, TrpC-Transient receptor potential channel C, Sema4a-Semaphorin 4a.
DISCI binds to PDE4D loci
60
We chose to further characterize the ATF4 binding site within the PDE4D gene. PDE4D
is one of four genes encoding a variety of cyclic adenosine monophosphate (cAMP)-specific,
rolipram-sensitive phosphodiesterases (Jin et al., 1998; Zhang et al., 1999; Houslay, 2010).
Animal models of PDE4D loss of function exhibit a phenotype similar to mice under
antidepressant treatment; they have a decreased immobility time in the forced swim test (FST)
(Zhang et al., 2002; Li et al., 2011 b; Schaefer et al., 2012) and have increased neurogenesis (Li
et al., 2011b). Furthermore, PDE4D activity is known to be directly modulated by DISC1 through
direct protein-protein interaction (Millar et al., 2005; Murdoch et al., 2007; Bradshaw et al., 2011).
To test our hypothesis that ATF4 mediated gene transcription is modulated by DISC1 via ATF4,
we conducted a ChlP-PCR using a polyclonal DISCI antibody targeted to the N-terminus of
DISC1, using samples from 8-week old wild-type (WT), ATF4 heterozygous (ATF4*') and ATF4
knockout (ATF4~'~) littermate mice. DISC1 binding to this locus is dependent on ATF4 in a
dosage-dependent manner (Figure 2A). Our results suggest that the association with DISC1
and the PDE4D loci is mediated by ATF4.
61
B.
A.
Scr
ATF4*'* ATF4*/- ATF4-'
PDE4D1
IgG
DISC1
-
m
e
PDE4D3
Input
Scr
-
PDE4D9
DISC-1 ab
ATF4
U am-4
PDE4D6
C.
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PDE4D8
ATF4--
ATF4 03
DISC-I
GADPH
D.
PDE4D9
# 4
GAPDH
a
ATF4*'*
ATF4*/-
C57/BL6
129B6
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O
-m -m
a
-ua
e
E.
PDE4D9
,
...
.
GAPDH %.w# %P*4 teve eoamw
"wo
Figure 2: DISC-I binds to ATF4 binding site within PDE4D region to regulate PDE4D9
expression. A. DISC-1 binding to region within PDE4D gene is ATF4 dependent. B. Infection of
cortico-hippocampal neurons with lentiviri expressing ATF4 or DISC1 shRNA results in the
62
specific increase of PDE4D9 transcripts. C. In situ hybridization of El 5 WT or ATF4-demonstrates elevated levels of PDE4D9 in the absence of ATF4. D. PDE4D9 protein levels are
elevated in 12-week old ATF4*'- mice compared to WT mice. E. mRNA levels of PDE4D9 are
increased in the 129/B6 congenic mouse model relative to C57/B6 littermates. 129 mice were
backcrossed over 10 generations, selecting for mice that retained the deletion within the DISC1
gene at each generation. RNA was isolated from forebrain and subjected to reverse
transcription, and the unique region of PDE4D9 was amplified from the cDNA. These mice also
display higher levels of PDE4D9.
DISCI and ATF4 both specifically regulate PDE4D9
The PDE4D gene locus encodes numerous variants of PDE4D, all of which share a
common catalytic domain, but are differentiated from each other by their unique N-terminal
fragment encoded by specific N-terminal exons that are thought to confer a definite subcellular
localization, and thus distinct functions, to each variant. We sought to determine which, if any, of
these variants were regulated by the ATF4-DISC1 binding site, using semiquantitative PCR with
primers previously optimized to determine relative PDE4D levels (Nemoz et al., 1996; Jin et al.,
1998; Richter et al., 2005). mRNA obtained from days in vitro (DIV) 14 cultured corticohippocampal neurons infected at DIV 4 with control, ATF4, or DISC1 shRNA expressing lentiviri
(Mao et al., 2009) revealed a marked and specific increase in the levels of PDE4D9 transcript,
but not others (Figure 2B). In situ hybridization using an antisense probe targeted to the unique
region of PDE4D9 at embryonic day 15 (E15; when ATF4 levels are at their peak) reveal that
there is a substantial increase in the levels of PDE4D9 transcript in ATF4-'- mice compared to
widtype littermates (Figure 2C). PDE4D9 protein levels are significantly upregulated in
hippocampal brain lysates from 8-week old ATF4'- mice compared to WT littermates (Figure
2D). Collectively, these results indicate that loss of ATF4 or DISC1 results in an increase in
PDE4D9 mRNA and protein levels. Thus, at baseline, ATF4 and DISC1 occupancy of the
PDE4D locus act as transcriptional repressors specifically for the PDE4D9 isoform.
63
To determine whether this increase in transcript can be exclusively accounted for by
transcriptional activity, and not by other factors such as altered transcript stability, we created
several luciferase constructs. We placed the 1000bp region immediately upstream of PDE4D9
coding region in front of the luciferase gene and compared it to the baseline activity of the pgl3luciferase construct, which does not have a promoter driving luciferase activity. The 1000bp
region upregulates luciferase activity indicating that this region acts as a promoter. We then
assayed whether the ATF4/DISC1 binding locus had any effect on the transcriptional activity by
the promoter by taking the ChIP region (-450bp), dividing it into four fragments, and putting
each fragment upstream of the 1000bp PDE4D9 promoter. The effect on transcriptional activity
for fragments A, B and C was minimal, while fragment D significantly altered the baseline
activity of the construct (Figure 3A,B); we chose fragment Dfor further evaluation. When we
subjected cells overexpressing this fragment to ATF4/ DISC1 knockdown, the luciferase activity
increased dramatically when exposed to DISC1 knockdown (Figure 3D). Moreover, when a
putative ATF4 binding site within this fragment was subsequently mutated, the ability of DISC1
shRNA to enhance transcription was diminished (Figure 3D). Together, these results indicate
that the ATF4/DISC1 binding site acts as an enhancer to the endogenous PDE4D9 promoter,
and that the loss of DISC1 significantly increases the transcriptional activity downstream of the
PDE4D9 promoter under the influence of this enhancer.
64
A.
D7
I
DIM 06010
0DO
DS
rim
l
k
U.
pGI3 (luc)
4D9pro
Enh4D9pro
4 pr
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C.
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900 800 U
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(5
I0
I.U
700 600 -
0
300-
4..
(U
0
--~
500400 -
Consensus CRE/CEBP
CHOP AARE
ASNS NSRE-1
EnhDPDE4D9
mutEnhDPDE4D9
.
,
.
.
.
.TGACGCAAT.
. TGATGAAAC.
.TGATGCAAT.
.GGGTGCAAT
.ACCTAGGTT.
2
2001000-
"IT AT
--
1
C,
N.S.
**
***
3000-
D.
E 2000C
1000"O
LL
shRNA
Sc+
ATF4 DISC1 -
+
+
-
+
+
mutEnhD 4D9proLuc
EnhD 4D9proLuc
4D9proLuc
+
-
+
-
-
-
-
-
-
+
+
+
+
+
+
-
+
+
-
-
+
+
+
-
+
+
+
65
2
3
4
5
QAAI
6
7
8
9
Figure 3: The ATF4-DISC1 binding region acts as a transcriptional repressor to
endogenous PDE4D9 promoter. A. Schematic of constructs used to study the transcriptional
effects of the ATF4-DISC1 binding region. B. Placement of the 1000bp region immediately
upstream of PDE4D9 coding region in front of the luciferase gene upregulates luciferase activity
indicating that this region acts as a promoter. Fragment D significantly altered luciferase activity
relative to the others (n=5 ANOVA p<0.0001. * Dunnett's test p<0.05 for comparison of luc to all
others, ***Tukey's test p<0.001 for EnhDPDE4D9 compared to other fragments, PDE4D9
promoter only). C. A putative CRE/CEBP binding site was identified within EnhD. The site was
mutated to eliminate sequence similarity to CRE/CEBP binding site. D. Comparison of the
effects of control, ATF4 or DISC1 shRNA combinations on luciferase activity on
EnhDPDE4D9The presence of enhancer fragment D (EnhD) upstream of the 4D9 promoter is
critical for the dramatic elevation in signal seen after ATF4, DISC1 shRNA expression (3E)(n=5
ANOVA p<0.0001, ***Tukey's test p<0.001 for comparisons between EnhDPDE4D9 DISC1
shRNA and DISC1/ATF4 shRNA compared to all others).
Cocaine and amphetamines are known to act through the excitation of dopaminergic
signaling: they are abused for their euphoric effects, and used in animals as a model for
psychosis. Amphetamine is also used in the treatment of psychiatric symptoms in attentiondeficit and hyperactivity disorder (ADHD), implicating the importance of their action on
psychopathology. ATF4-dependent transcription is known to be mediated by several factors,
including oxidative/nutrient stress, and cAMP-induced increases in PKA activity; notably, CART,
or cocaine-amphetamine response transcript, is a product of ATF4 mediated transcription
known to be upregulated upon treatment with cocaine and amphetamines. This led us to
hypothesize whether PDE4D9 levels may also be regulated by similar mechanisms. Treatment
with dopamine (100um) as well as isoproterenol (100um), a beta-adrenergic receptor agonist,
but not potassium chloride (KCL, 55mM), which induces generalized depolarization, or
Prostaglandin E, (PGE, 10mM, not shown) which has been reported to increase
phosphodiesterase activity in other systems, in DIV14 cultured cortico-hippocampal neurons
lead to increases in PDE4D levels as assayed by observable changes in mRNA levels by semiquantitative qPCR (Figure 4A). These results suggested that agonists of D1-type or Gas coupled
G-protein coupled receptor stimulation lead to increases in PDE4D9. We further validated this
hypothesis by using qPCR to assay for changes in PDE4D9 levels, comparing vehicle,
66
dopamine (10um), D1-type receptor agonist SKF81297 (3uM), and D2-type receptor agonist
quinpirole (1OuM) treatment. Both dopamine and SKF81297, but not quinpirole, lead to
elevations of PDE4D9 transcript, indicating that D1-type receptor stimulation increases levels of
PDE4D9 (Figure 4B). Amphetamine treatment (0.35mg/kg) of 8-week old mice resulted in
elevated PDE4D9 mRNA levels as well as detectable changes in PDE4D9 protein in mouse
hippocampus (Figure 4C, D). These results indicate that PDE4D9 levels are mediated
physiologically by dopamine via a D1-type receptor signaling mechanism, and that
neuropharmacologically relevant doses of amphetamine can increase levels of PDE4D9.
67
A.
Veh
2h
Figure 4
B.
4h
PDE4D9 0
N.S
GAPDH
3--
Dopamine (100pM)
Veh
7h
Ah
2-
PDE4D9
-r-
GAPDH
Isoproternol (100pM)
0
Veh
9h
Vehicle
Ah
PDE4D9
Dopamine SKF81297 Quinpirole
(10pM)
(3pM)
(10pM)
GAPDH
KCI (55mM)
C.
Veh
D.
4h
*
PDE4D9
1.6
GAPDH OWnO
1.2
1
0.8-
0
U-
E.
Veh
2h
T
0.4
0
4h
Vehicle
Amphetamine
PDE4D9
GAPDH
Figure 4: G,-coupled G-protein coupled receptor signaling leads to increases in PDE4D9
levels. A. Dopamine and Isoproternol (P-adrenergic receptor agonist), but not KCI (general
activator of neuronal activity via depolarization), leads to increases in PDE4D9 transcript. B. D168
type, but not D2-type dopamine receptor agonists, lead to detectable changes in PDE4D9
transcript. Dopamine (10pM), SKF81297 (3pM), but not quinpirole (10pM) treatment lead to
detectable increases in PDE4D9 levels, assayed by qPCR for unique region of PDE4D9.N=3
ANOVA p<0.05, *p<0.05 Dunnett's test. C. 0.35mg/kg amphetamine treatment of C57B6/J mice
leads to detectable increases in PDE4D9 transcript. N=4, student's T-test p<0.05). D.
0.35mg/kg amphetamine treatment leads to detectable increases in PDE4D9 protein levels.
To examine the role of DISC1 and ATF4 in the aforementioned phenomenon, we
conducted the ChIP experiments described previously in mice treated either with amphetamine
(0.35mg/kg) or with vehicle. PDE4D loci occupancy for DISC1 was significantly diminished
either two or four hours after treatment, compared to vehicle or untreated mice (Figure 5A);
vehicle treatment at these time-points, were similar to untreated mice (data not shown).
Interestingly, we observed a detectable, but non-statistically significant, increase in the levels of
ATF4 occupancy at this locus with this treatment (Figure 4E). Increases in the levels of ATF4 in
the hippocampus after amphetamine treatment have been reported elsewhere in the literature
(Green et al., 2008). Taken together, our results indicate that PDE4D9 levels are repressed by
the interaction between ATF4, DISC1 and the PDE4D loci, and that the dopaminergic
stimulation of D1 type receptors leads to the dissociation of DISC1 from the loci, leading to
increases in PDE4D9 transcription.
69
A.
0
2h
B.
4h
DISC1
DISC1
E 2.
C
ATF4
'S 1-
IgG
0-
Input Ow q
Oh
D.
2h
C.
0.
CL
C
0
CL
x
CL
H-
C
0
0
z
ATF4
1.5-
T
1.0-
GSTATF4
C
a8
C
GEPDISCI
0.50-
Oh
E.
F.
U
2
(0
C~)
N-
H
4h
LL
x~.u
CO,
CO
DISC1
0 1.50tU
0
NC,)
2h
Enzyme Treatment
1.75-
C
4h
*
*
1.251.000.75-
L0 0.50-
0.250.00CIP
No Tx
PKA
Figure 5: Gs-Coupled and downstream effects lead to dissociation of DISC1 from ATF4.
A. ChIP using DISC1 and ATF4 antibodies after amphetamine treatment (0.35mg/kg) from
C57/BI6J hippocampus reveals that there is loss occupancy of DISC1 at the PDE4D9 locus
compared to vehicle injected mice. B. Quantification of DISC1 occupancy at PDE4D9 locus.
(n=6, ANOVA p<0.05, Dunnett's test p<0.001 for comparisons between vehicle injection and 2
and 4 hours after amphetamine injection). C. Quantification of ATF4 occupancy at the PDE4D9
locus. ANOVA p=0.13, n=6) (D)Purified GST-tagged ATF4 protein and GFP-DISC1 protein,
from overexpression in bacteria and HEK 293T cells, respectively, decrease interaction in vitro
70
after the
addition of the catalytic unit of PKA. E. Quantification of (D). N=4, ANOVA p<0.05,
*p<0.0 5 , **p<0.01 by Neuman-Kewls post hoc analysis.
Both ATF4 and DISC1 have known PKA phosphorylation sites (Elefteriou et al., 2005;
Ishizuka et al., 2011). ATF4 has been shown to turn from a repressor to a transcriptional
activator after PKA phosphorylation (Elefteriou et al., 2005), and DISC1 localization has been
reported to be altered by PKA phosphorylation (Ishizuka et al., 2011). We sought to determine
whether phosphorylation sites have an effect on DISC1-ATF4 interaction. To do this, full-length
GFP-tagged DISC1 and GST-ATF4 proteins were purified and recombined in an in vitro context.
The DISCI and ATF4 interaction was substantially diminished upon the addition of the catalytic
subunit of PKA, which was not observed with the addition of equimolar concentrations of BSA;
the addition of alkaline phosphatase increased the observed interaction, compared to BSA
control (Figure 5F). Neither the mutation of the DISC1 S710 site, nor the ATF4 S254 site
resulted in significant changes in DISC-1 ATF4 interaction (data not shown). Also, contrary to
previously published results (Malavasi et al., 2012), the ATF4/ DISC1 interaction did not seem
to be significantly altered between L607 and 607F DISC1 proteins (data not shown). Our results
suggest that in response to Gs coupled GPCR stimulation DISC1 leaves from the ATF4/DISC1
binding locus, and that this effect is likely mediated through direct phosphorylation events on
ATF4 and/ or DISC1 by PKA.
DISCUSSION
The DISC1 gene has been clearly implicated in the manifestation of psychiatric disorder
phenotypes. Here we uncover a novel transcriptional mechanism by which DISCI regulates 1
specific variant of one of the four genes that encode the rolipram-sensitive, cAMP specific
phosphodiesterases, genes that have been implicated in psychiatric disorder phenotype and
antidepressant efficacy.
71
DISC1 binds to and represses an ATF4 transcriptional target
Here we report that DISC1 is, in fact, bound to mammalian ATF4 targets in a manner
that is dependent on ATF4. This is an important result because previous work has been
conducted under the assumption that this isthe case (Sawamura et al., 2008; Malavasi et al.,
2012). Our finding that endogenous DISC1, immunoprecipitated by antibodies that bind directly
to DISC1 can be found at ATF4 regulated loci, gives much credence to the work conducted
using transfected luciferase constructs that contain canonical CRE sites as readout of DISC1
function. Our data suggests that mutating a putative CRE/CEBP site renders the binding site
null of its enhancer function, which further adds to the literature that has documented similar
sites as sites of ATF4 action (Siu et al., 2002; Fujita et al., 2007; Gombart et al., 2007). It is
tempting to speculate that endogenous ATF4 prefers to bind to this type of genetic site, rather
than the traditionally reported CRE site. Our results also indicate that, in addition to ATF4
interaction, DISC1 has other mechanisms by which it regulates transcriptional activity, because
DISC1 lacks any enzymatic activity. The role of DISC1 interaction with other transcriptionally
relevant partners, such as the SWI/SNF proteins, is lacking in the literature and further studies
in this area would add to our understanding of DISC1 function in the modulation of transcription.
DISC1 ATF4 interaction is physiologically regulated
Interestingly, numerous psychotherapeutic agents impact cAMP levels via secondary
actions. Amphetamine result in increased dopaminergic release and is used in the treatment of
ADHD. Improper administration leads to euphoria. MAOi's are classical antidepressants and act
by elevating dopamine, serotonin and norepinephrine. New-generation antidepressants elevate
cAMP via increases in 5-HT and norepinephrine via selective serotonin or serotoninnorepinephrine reuptake inhibition (SSRIs and SNRIs). Our study provides further clues into the
development of psychiatric phenotypes secondary to the dysregulation of this pathway. An
72
increase in PDE4D9 secondary to the activation of cAMP, is a form of transcriptional feedback
regulation, one normally predicted to be necessary for the proper maintenance of baseline
cAMP levels and the maintenance of activity (Figure 6). Our results indicate that, in the absence
of DISC1, this feedback loop would be not just inactivated, but rather usurped. Under DISCI
knockdown conditions cells already have a higher baseline level of PDE4D9, which is normally
increased secondary to Gs coupled dopamine D1-type and beta-adrenergic receptor stimulation.
A baseline increase in phosphodiesterases would lead to perturbed signal propagation
downstream of these receptors (Figure 6). This is in addition to its previously reported direct
interaction and inhibition of its catalytic activity, which has shown that DISC1 is able to regulate
some, but not all, gene products of the four PDE4 genes in an activity dependent manner (Millar
et al., 2005; Bradshaw et al., 2011).
There is evidence suggesting that PDE4D levels are regulated by both chronic
antidepressants and rolipram administration, further implicating the cAMP pathway in the
regulation of this genetic locus. Our study provides a biochemical basis for this regulation. A
previous study reported that chronic antidepressant or rolipram treatment resulted in the
increase of PDE4D3 variant, based on the band size of the increased PDE4D variant(DIaboga
et al., 2006). This study was published just after the identification of PDE4D9. Another study
which characterized the expression of the different PDE4D's, noted that tissues previously
thought to express predominantly PDE4D3, in fact expressed three PDE4D's of very close
molecular size, PDE4D3, PDE4D8 and PDE4D9 (Richter et al., 2005). It is likely that the study
from Dlaboga and colleagues had documented the increase in PDE4D9 but miscategorized it as
PDE4D3.
73
Figure 6
Baseline
A.
G-proteln ;Ps)
.
B.
cAMP = 0
0
e
en
Dr
0
D1D2DOMi
Activation of Gs-GPCR
cAMP= 0
C.
DISC-i dysfunction-mediated depression
Neurotransmitter
0
cAMP =0
G-protein(Gs)
PDE4D9 levels are high at ba eline,
inhibiting neurotransmittermediated activation of cAMP->PKA
-ATF4-mediated transcription ishigher
-CREB-mediated transcription is lower
Dr
DeD
DURDIM9
74
Figure 6: Model of endogenous DISC1/ATF4 function at the PDE4D binding site, at
baseline (A), after Gs-coupled G-protein coupled receptor stimulation (B), or in DISC1 loss of
function (C).
Our results may also offer interesting insights on further therapeutic approaches
targeting PDE4's in the treatment of psychiatric disorders. DISC1 disrupts cAMP signaling by
perturbing the relative levels of the PDE4 variants, in addition to selectively affecting the activity
of some, but not all of the PDE4 variants. The perturbation of this pathway may disrupt the
ability of neurons to respond appropriately to neurotransmitter signaling, ultimately affecting the
neuronal circuitry and resulting in abnormal behavioral responses that may manifest as
psychiatric disease.
Our lab has previously demonstrated that DISC1 effects on behavior can be rescued by
the use of GSK3p inhibitors. The current finding is consistent with previous findings that a loss
of DISC1 function would lead to increased phosphodiesterase activity. Further experiments are
ongoing to determine if there will be synergistic effects of the combined use of GSK3p and
PDE4 inhibitors on depressive phenotypes as well as their effect on neurogenesis. Preliminary
results, as well as evidence from previous literature suggest that this is indeed the case (Zhang
et al., 2006b; Taurin et al., 2008).
Methods
Generation of antibodies
Polyclonal antibodies against the unique portion of PDE4D9 was raised by the generation of
a peptide identical to the unique sequence of PDE4D9(PDE4D9unq), amino acid sequence
MSIIMKPRSRSTSSLRTTEAVC. Briefly, the peptide was generated and purified by HPLC,
then 500 pg was injected, along with Freund's complete adjuvant (FCA) into 2 rabbits after
prebleeding. The rabbits received a booster dose of 500pg PDE4D9unq and Freund's
75
Incomplete adjuvant (FIA) 20 days after initial injection. 4 additional booster doses of 250pg
PDE4D9unq and FIA were given at 20 day intervals. At 52 days post initial injection, a test
bleed was obtained to assay antibody titer. Two production bleeds were obtained at 94 and
118 days after initial antigen injection. Sera obtained from the bleeds were then subjected to
affinity column purification to obtain purified antibodies. Antibodies used for this study were
from the first bleed. All in vivo work was done by Covance Inc. in accordance with MIT CAC
guidelines. Antibody purification from sera was done in Tsai lab.
Polyclonal antibodies against the N-terminal portion of DISC-1 was generated in a protocol
similar to that described above, save for the dose of injected antigen: 250pg DISC-1 N-term
was initially injected, followed by 125pg booster doses at 20 day intervals. 6-His-DISC-1 Nterm was generated by cloning the N-terminal portion of mouse DISC1, amino acids 1-220
into the pET-32b+ vector. The plasmid was then transformed into BL21 (DE3) Competent
E.Coli. Protein expression was driven by IPTG stimulation. The protein was purified using
column affinity purification, and the his tag was removed by thrombin cleavage by the addition
of 1 unit of thrombin per column overnight. The supernatant was collected and delivered to
Covance for in vivo work.
Commercial Antibodies used for the study
Antibody
Source
Dilution
ATF-4 (C-20)
GAPDH(6C2)
FLAG M2
(mouse)
GFP (Rb)
Santa Cruz
Santa Cruz
2.5pg per sample ChIP
1:5000
Sigma-Aldrich
Injitrogen
1:2000 for western, 2pg per sample IP
1:2000
Myc tag
Cell Signaling Technology
1:1000, 2ig per sample IP
Compounds used for the study
Name
KCI
Concentration
55mM
76
Source
Sigma-Aldrich
Forskolin
Dopamine
l0pM
Variable
Tocris
Sigma-Aldrich
SKF81297
Quinpirole
3lM
10p1M
Sigma-Aldrich
Tocris
Prostaglandin E
Isoproternol
Tocris
Sigma-Aldrich
Protein Kinase A, catalytic subunit
loM
100pM
2500
units/reaction
D-Amphetamine
0.35mg/kg
poly-D-lysine
50pg/ml
Laminin
Trypsin
BSA
PKA, catalytic subunit
Calf Alkaline Phosphatase (CIP)
30pg/ml
0.25%
Variable
2500000units/ml
10,000 units/mI
NEB
Tocris
BD life
sciences
BD life
Sciences
Gibco
Invitrogen
NEB
NEB
Primers used for the study
NAME OF
TRANSCRIPT
PDE4D1/2 RT
PDE4D3 RT
PDE4D6 RT
FORWARD
TATGAAGGAGCAGCCCTCATG
CAGAAGGCATTCCTGGATATG
CACATTTTAGAACTTGCTGTCAC
REVERSE
CCAGGACATCTTCCTGCTCTG
GCCACAAGTGCCTCTTGCAGC
ATGAGCATTATTATGAAGCCG
TGGCCAGTTTCTGGTAGGCCTC
CCAGGACATCTTCCTGCTCTG
TCCAGACACCAGTCCAGCTCCTCC
A
TGGCCAGTTTCTGGTAGGCCTC
ATF4 RT
GGAATGGCCGGCTATGG
TCCCGGAAAAGGCATCCT
DISC-1 RT
TTGCTGGAAGCCAAGATGCTGG
CTTCACGCCTATGGCTTCGC
GAPDH RT
TCACCACCATGGAGAAGGC
PDE4D8 RI
PDE4D9 RT
3XMYCPDE4D1
GCCACCATGGAGAGGGACACTTG
TG
3XMYCPDE4D2
GCCACCATGGCCTCCAACAAGTT
3XMYCPDE4D5
GCCACCATGGCTCAGCAGACGAC
AAGC
3XMYCPDE4D6
GCCACCATGCCTGAAGCAAACTA
TTTATTG
3XMYCPDE4D7
GCCACCATGCTCAAACCAGAGTG
TTGGGAT
3XMYCPDE4D9
GCCACCATGAGCATTATTATGAA
GCCG
3XMYCPDE4D3
GCCACCATGGAGGCAGAGGGCA
GCAGCGTG
GCTAAGCAGTTGGTGGTGCA
TTACGTATCAGGACAGCAGTCATCC
CTAGGGCG
TTACGTATCAGGACAGCAGTCATCC
CTAGGGCG
TTACGTATCAGGACAGCAGTCATCC
CTAGGGCG
TTACGTATCAGGACAGCAGTCATCC
CTAGGGCG
TTACGTATCAGGACAGCAGTCATCC
CTAGGGCG
TTACGTATCAGGACAGCAGTCATCC
CTAGGGCG
TTACGTATCAGGACAGCAGTCATCC
CTAGGGCG
CAATACCGAATGCCCCTTTA
CAGAGTGTGGCAAACAGGAA
4D CHIP
77
4D9
PROMOTOR
4D9 ENHANCER
FRAGMENT A
4D9 ENHANCER
FRAGMENT B
4D9 ENHANCER
FRAGMENT C
4D9 ENHANCER
FRAGMENT D
A4D9
ENHANCER
FRAGMENT D
CONTROL
SHRNA1
DISC1SHRNA1
DISC1SHRNA2
ATF4SHRNA1
ATF4SHRNA2
GCGCTCGAGCTGTATAATGGAAA
TGACTTTCTG
TACATACGTACCTTATATCATGGC
ACTGAAGTACTTTACCCCACACCT
ATGTGTACTTAACATCCCTGTACT
TAGCAGCAACTCTGTAGCTGTAC
AACGTTAGTTTCTTGGG
AACCTTTGCCATGGGCCAATACC
GAATGCCCCTTTATTATTGAGTAA
ACCTGCATCTGGCCTTCCCCCAT
CGCTGCAGACTAATCTTACCCTTT
GTTCGCCTCGTCCATGTTT
GGTATGGTTATAGACTCTCTGTGT
ATCTCTTGGAGGGGACACAAACA
ATGGACGCTGTTTGTCTTTTCCTG
TTTGCCACACTCTGTGGAGTTCTT
GACACACTCTAGAGGGTGCAATA
GCATTCTGTAATATTGTGGTCCAA
TCACGTTTCTTCATACTCTGAATG
ATACTACATTGAATTGGTTTTTCC
ACGGCCTG
GACACACTCTAGAACCTAGGTTG
AGCATTCTGTAATATTGTGGTCCA
ATCACGTTTCTTCATACTCTGAAT
GATACTACATTGAATTGGTTTTTC
CACGGCCTG
TCGGCTGAAACAAGAGTTGGTTC
AAGAGACCAACTCTTGTTTCAGC
CGCTTTTTTC
TGGCAAACACTGTGAAGTGCTTC
AAGAGAGCACTTCACAGTGTTTG
CCTTTTTTC
TGCAGGAGGTCAGCAAGGCCTTG
TTCAAGAGACAAGGCCTTGCTGA
CCTCCTGC TTTTTTC
TGAGCATTCCTTTAGTTTAGTTCA
AGAGACTAAACTAAAGGAATGCT
CTTTTTTC
TGAGAAGAGAGTTCCGTAATATTC
AAGAGATATTACGGAACTCTCTTC
TG TTTTTTC
PDE4D9
PDE4D9
SHRNA1
TGCCAAGATCCAGGTCTACATTC
AAGAGATGTAGACCTGGATCTTG
GCTTTTTTC
PDE4D9
PDE4D9
SHRNA2
TGTCCGTTGTTATGGAATTGTTCA
AGAGACAATTCCATAACAACGGA
CTTTTTTC
GCGAGATCTGGGAAAGGGAAATGT
TCAAGTCTTCTGATC
CCCAAGAAACTAACGTTGTACAGCT
ACAGAGTTGCTGCTAAGTACAGGG
ATGTTAAGTACACATAGGTGTGGG
GTAAAGTACTTCAGTGCCATGATAT
AAGGTACGTATGTA
AAACATGGACGAGGCGAACAAAGG
GTAAGATTAGTCTGCAGCGATGGG
GGAAGGCCAGATGCAGGTTTACTC
AATAATAAAGGGGCATTCGGTATTG
GCCCATGGCAAAGGTT
AAGAACTCCACAGAGTGTGGCAAA
CAGGAAAAGACAAACAGCGTCCAT
TGTTTGTGTCCCCTCCAAGAGATAC
ACAGAGAGTCTATAACCATACC
CAGGCCGTGGAAAAACCAATTCAAT
GTAGTATCATTCAGAGTATGAAGAA
ACGTGATTGGACCACAATATTACAG
AATGCTATTGCACCCTCTAGAGTGT
GTC
CAGGCCGTGGAAAAACCAATTCAAT
GTAGTATCATTCAGAGTATGAAGAA
ACGTGATTGGACCACAATATTACAG
AATGCTCAACCTAGGTTCTAGAGTG
TGTC
TCGAGAAAAAACGGCTGAAACAAG
AGTTGGTTCAAGAGACCAACTCTTG
TTTCAGCCGCA
TCGAGAAAAAAGGCAAACACTGTG
AAGTGCTCTCTTGAAGCACTTCACA
GTGTTTGCCA
TCGAGAAAAAAGCAGGAGGTCAGC
AAGGCCTTGTCTCTTGAACAAGGC
CTTGCTGACCTCCTGCA
TCGAGAAAAAAGAGCATTCCTTTAG
TTTAGTCTCTTGAACTAAACTAAAG
GAATGCTCA
TCGAGAAAAAAGAGAAGAGAGTTC
CGTAATATCTCTTGAATATTACGGA
ACTCTCTTCTGA
TCGAGAAAAAAGCCAAGATCCAGG
TCTACATCTCTTGAATGTAGACCTG
GATCTTGGCA
TCGAGAAAAAAGTCCGTTGTTATGG
AATTGTCTCTTGAACAATTCCATAA
CAACGGACA
4D primer sequences adapted from Plasmids used for the study adapted from (Richter et al.,
2005) to account for mouse, not rat, sequences
78
Plasmids used in study
Name
3xFLAG vector
control
3x-FLAG ATF4
pEGFP-C1
GFPmDISC1-FI
GFP-mDISC1
S710A
GFP-mDISC1
T736A
GFP-mDISC1
S744A
GFP-hDISC1
WT
GFP-hDISC1
S704C
GFP-hDISC1
L607F
pGEX-4T2
GST-mATF4
pRK5 HAmDISC1
pRK5 HAmDISC1 S710A
pRK5 HAmDISC1 S710E
p113.7
pil3.7mCherry Scr
p113.7mCherryDISCIN1
p113.7mCherryDISC1N8
p113.7-ATF4-2
pil3.7-ATF4-4
Vector
Tag
Expression (promotor)
mDISC1-T1
pcDNA3.1
pcDNA3.1
pEGFP-C1
pEGFP-C1
3x-FLAG
3x-FLAG
GFP
GFP
Mammalian
Mammalian
Mammalian
Mammalian
mDISC1-T1
pEGFP-C1
GFP
Mammalian (CMV)
mDISC1-T1
pEGFP-C1
GFP
Mammalian (CMV)
mDISC1-T1
pEGFP-C1
GFP
Mammalian (CMV)
hDISC1
pEGFP-C1
Mammalian (CMV)
hDISC1
pEGFP-C1
Mammalian (CMV)
hDISC1
pEGFP-C1
GST
mATF4
pGEX-4T2
pGEX-4T2
Mammalian (CMV)
Bacterial (Ptac)
GST
Bacterial (Ptac)
mDISC1
pRK5
HA
Mammalian (CMV)
mDISC1
pRK5
HA
Mammalian (CMV)
mDISC1
pRK5
HA
GFP(tag)
p113.7
GFP
mCherry(tag)
p113.7mcherry
mCherry
DISC1shRNA1
p113.7mCherry
mCherry
p113.7mCherry
p113.7
mCherry
GFP
Mammalian (CMV)
Mammalian (CMV for
GFP, U6 for shRNA),
lentiviral
Mammalian (CMV for
GFP, U6 for shRNA),
lentiviral
Mammalian (CMV for
GFP, U6 for shRNA),
lentiviral
Mammalian (CMV for
GFP, U6 for shRNA),
lentiviral
p113.7
GFP
Insert
mATF4
DISC1shRNA2
ATF4shRNA2
ATF4shRNA4
79
(CMV)
(CMV)
(CMV)
(CMV)
pGl3
pG13
Luciferase
None
1013bp upstream of
PDE4D9 transcription
initiation
pGl3
Luciferase
4D9 promoter
Enhancer region A
pG13
Luciferase
4D9 promoter, Enh A
Enhancer region B
pGl3
Luciferase
4D9 promoter, Enh B
Enhancer region C
pGl3
Luciferase
4D9 promoter, Enh C
4D9pro-luc
EnhA4D9proluc
EnhB4D9proluc
EnhC4D9proluc
EnhD4D9proluc
AEnhD4D9proluc
Enhancer region D
pGl3
Luciferase
4D9 promoter, Enh D
mutant Enhancer region D
pGl3
Luciferase
HSVLT-4D9
mPDE4D9
HSVLT
IRES-GFP
HSVLT-GFP
TK-Renilla
Luciferase
pSP6T7
pSP64D9uniqueT7
GFP
HSVLT
Renilla Luciferase
TKRL
pSP6T7
IRES-GFP
Renilla
Luciferase
4D9 promoter, mutEnh D
Mammalian, Proprietary
(Dr.Rachel Neve)
Mammalian, Proprietary
(Dr.Rachel Neve)
Mammalian, Constitutive
(Thymidine Kinase)
In Vitro
4D9 unique
pSP6T7
In Vitro
GFP-mDISC1 and 3xFLAG-mDISC1 has been described previously (Mao et al., 2009). Briefly,
the full-length human DISC1 transcript was isolated from cDNA, then subcloned into the
pEGFP-C1 vector (Clontech, Takara). S710A, T736A, S744A mutants were generated by sitedirected mutagenesis using the quickchange lightning site directed mutagenesis kit. HAmDISClwt, S710A, S710E, were kind gifts from Dr. Ishizuka. GST-ATF4 has been described
previously: full length mATF4 was cloned into the pGEX-4T-2 vector. 3x-myc
PDE4D2,3,5,6,7,8, and 9 were generated by PCR amplification of the full-length cDNA fragment
from a cDNA library generated from RNA extraction and cDNA synthesis, as described below,
using 5' primers flanked by a Kozak (GCCACCATG) sequence and 3' primers flanked by a Xhol
restriction site, and then cloned into a pcDNA(3.1)+ vector modified to add 3 myc tags at the Cterminal end of the insert. The vector was digested using EcoRV and Xhol, and the inserts
were annealed to the vector.
80
Production of plentilox3.7 ATF4 shRNA
ATF4 and DISC1 shRNA was cloned into the plentilox 3.7 vector (Addgene plasmid 11795) as
previously described (Mao et al., 2009). The DISC1 shRNA sequences used are the same as
those described. Briefly, complimentary 5' phosphorylated oligonucleotides for ATF4, DISC1,
and PDE4D9, provided above, were annealed, digested with Xhol and ligated into the plentilox
3.7 vector that had been digested with Hpal and Xhol. Proper insertion and orientation of the
sequence downstream of the U6 promoter was confirmed using a sequencing primer with the
sequence cagtgcaggggaaagaatagtagac. To create DISC1shRNA and scr-shRNA mCherry, the
GFP cDNA sequence contained within p113.7 was excised and replaced with the mCherry cDNA
sequence, obtained by amplification using from pRK5-mCherry (addgene).
Chromatin Immunoprecipitation
ChIP was conducted from either neurons isolated from adult (12 week-old) hippocampus or
cortex by loose dounce, by modifying the conditions provided in the EZ-MagnaChlP kit
(Millipore). Briefly, cells were isolated by loose dounce, then crosslinked for 10 minutes in 1ml
1% formaldehyde-PBS with nutation in a 1.5ml eppendorf tube. Formaldehyde was then
quenched for 5 minutes by the addition of glycine, accompanied by nutation. Cells were
washed twice with ice-cold PBS, then, after removal of supernatant, lysed using 1mL ice-cold
cell lysis buffer with protease inhibitors. The supernatant was removed, and 0.5mL nuclear
lysis buffer was added to the cell pellet to lyse the nucleus. The lysate was sonicated to shear
the crosslinked DNA in conditions optimized to obtain -500bp fragments using a Branson 250-D
sonifier. Insoluble cell debris was spun down at 15,000xg. 50pL lysate was used for each
subsequent reaction. Lysate was diluted into 450pL dilution buffer, then 5pg of antibody or
Normal Rabit lgG and 20pL suspended protein A magnetic beads were added to the diluted
lysate. The mix was then incubated for 2 hours at 4 0C with rotation. Magnetic beads were
isolated, then washed twice with low salt immune complex wash buffer, twice with high salt
81
immune complex wash buffer, once with LiCI immune complex wash buffer, and once with TE
buffer. DNA was isolated from the beads by adding 100pl ChIP elution buffer with 1pl
proteinase K, and incubating for 2 hours at 62 degrees in a shaking incubator. Proteinase K
was inactivated by incubation at 950C for 10 minutes. The beads were removed, and 0.5ml bind
reagent A was added to each DNA sample. The mixture was then put through a spin filter in a
collection tube, and the spin filter was further washed wish Wash reagent B. Purified DNA was
eluted from the spin filter column with the addition of 3 0pl Elution buffer C. For all subsequent
reactions, 1pl of DNA was used for analysis.
Amphetamine treatment
Animals were injected with 0.35 mg/kg Amphetamine resuspended in 0.9% saline at 70ng/ul, or
with saline only in accordance with CAC procedures, then placed in a novel cage for 2 or 4
hours before being sacrificed by C02 euthanasia, followed by rapid isolation and processing of
brain samples. Briefly, after animals were sacrificed, the head was quickly dissected and rapidly
frozen by placement for 20 seconds in liquid nitrogen. Following rapid freezing, brains were
dissected out and separated by subregions on ice. Brains remained frozen in liquid nitrogen
until enough samples had been collected for parallel processing for Chromatin IP,protein, and
RNA isolation.
Primary cortico-hippocampal cultures
Cortico-hippocampal cultures were prepared from embryonic E15-16 swiss-webster
mice. Hippocampi and cortices were dissected, trypsinized, and plated in cell culture
plates coated with poly-D-lysine and laminin (BD Biosciences) at a density of 5.0 x 105
cells/ml in neurobasal media (Gibco/Invitrogen) containing 10% horse serum
(Invitrogen), penicillin/streptomycin (Invitrogen), and glutamax (Invitrogen). Plating media was
exchanged for neuronal media (neurobasal media supplemented with B27) 2-8 hours after
plating. Experiments were performed on 10 to 17 days in vitro (DIV) primary cultures.
82
Lentiviral mediated knockdown of targets.
Production and titration of virus.
Lentiviral particles were made as previously described 51. Briefly, HEK-293Tcells were plated
on 10 cm dishes in 10% FBS-DMEM and transfected at 95% confluency with 18 pg plenti 3.7,
6ug pCMV-AR8.9, and 5 pg pCMV-VSV-G per dish. The culture medium was switched to 30%
FBS DMEM 12 hours after
transfection and viral supernatant was collected 48 and 96 hours later. Viral supernatant was
filtered through a 0.45pm cellular acetate vacuum filter (Corning 431155), and concentrated by
ultracentrifugation at 25,000 x g for 90 minutes. Viral pellets were resuspended in DPBS+0.1%
glucose and stored at -80 'C.
Viral titers were determined on HEK-293T cells plated at 2x1 05 cells/well in 6-well plates, and
serial dilutions of 1:200, 1:2000, and 1:20,000 were used to determine viral titer. After 48 hours
of viral supernatant application, % infected cells were determined by determining % fluorescent
cells/total # cells by visual inspection. Four fields of view were counted per well, and three wells
were inspected per dilution. Infection was performed with MOI =10.
mRNA isolation, analysis
RNA was isolated using the RNEasy plus mini kit. Briefly, neurons were isolated by either
aspiration of the media in the case of cultures, or by isolation, in the case of brain tissue. Buffer
RLT plus, supplanted with 1OpI p-mercaptoethanol/1ml was added to the cells, and the lysate
was homogenized using a 28 gauge needle and syringe. Genomic DNA was cleared from the
solution by passage through a gDNA binding column. RNA was isolated by passing the gDNA
cleared lysate through a RNA spin column after the addition of equal part 70% ethanol. The
bound RNA was purified with a series of wash steps: once with 700pl buffer RW1 and twice
83
with 500pI buffer RPE before isolation of RNA from the column using 30pl RNAse-free ddH 20.
Subsequent analysis was done using 1pi of isolated RNA per reaction.
cDNA synthesis, Semiquantitative PCR, quantitative PCR
equivalent amounts of RNA was used for cDNA synthesis using the first-strand cDNA synthesis
kit (Invitrogen). Briefly, RNA concentration was measured and 0.8ug total RNA was used per
reaction. The RNA was diluted to 8ul, and 1ul 50pM oligo dT and 1pl 10mM dNTP mix was
added. The reaction was incubated at 65 0 C for 5 minutes, then placed on ice. 2p 1Ox RT
buffer,4pl 25mM MgCl 2 , 2pl 100mM DTT, 1pI RNaseOUT (40U/pl), and 1pI Superscript Ill
reverse transcriptase per reaction was premixed, and lOpI of the mixture was added to each
RNA/primer/dNTP mix. The mixture was incubated for 50 minutes at 500C, and the reaction
was terminated by incubating at 850C for 5 minutes. 1pl RNase H was added to all mixes, and
incubated at 370C for 20 minutes. 1pi cDNA synthesis reaction was used for subsequent
analysis.
Semiquantitative PCR was conducted using the primers described above, at optimized
temperatures. Briefly, 1pi cDNA was added into a mixture of 9pl dnase-free H2 0 and 10pl 2x
PCR mastermix, a mixture of TAQ DNA polymerase and optimized enzyme buffer. Reactions
were terminated at 20 cycles for GAPDH, and multiple cycles were collected for other primers:
the cycle indicating the biggest difference between conditions was used for data collection: this
was typically between 25-32 cycles.
Quantitative PCR was conducted using the primers described above, at optimized temperatures.
Briefly, 1pl cDNA was added into 9pl dnase-free H2 0 and 10pl SsoFast Evagreen Supermix
(Bio-Rad). The qpcr optical reaction was conducted in a T100 thermal cycler fitted with a
CFX96 Touch real-time PCR system (Bio-Rad). All experiments were conducted with triplicated
samples for each cDNA sample per primer pair. All comparators, as well as loading controls
84
were loaded onto the same 96-well and normalization was conducted within each plate before
statistical analysis.
Luciferase Activity Assay
CAD or N2A cells were plated at a density of 2.5 x 1QA5 /ml in 24 well plates one day prior to
transfection with a combination of a plasmid expressing renilla luciferase under the control of a
human thymidine kinase 1 promotor (TK-RI, addgene), firefly luciferase within the pgl3
backbone under the control of numerous promotors and enhancers, as described, at a ratio of
1:10. For assays comparing ATF4 and DISC1 knockdown, they were also infected at an MOI=5
with lentivirus that transduce expression of shRNA driven by a mouse U6 promotor 2 hours after
transfection. Assays were conducted two days post transfection/viral transduction using the
dual-luciferase reporter assay (Promega) in conjunction with the Spectramax-L luminescence
microplate reader (Molecular Devices). Briefly, an hour before assaying, cells were lysed in
100pi 1x passive lysis buffer and rocked at room temperature for 20 minutes. Following cell
lysis, 30ul of each lysate was loaded onto a 96-well plate. 25ul of LARII was dispensed onto
each lysate to measure the firefly luciferase activity, followed by 25ul of Stop & Glo Reagent to
measure the Renilla luciferase activity. The ratio of firefly:renilla activity was then determined to
normalize the value of firefly luciferase activity to cell viability and transfection efficiency, as
assessed by the constitutive expression of renilla. A minimum of three wells' values were then
averaged to count as the normalized value for the experiment. This was further normalized to
the values of the control conditions, present on each plate, before combining them for statistical
analysis.
Immunoblot Analysis
Unless otherwise indicated, at the end of the indicated treatments, neurons or cells were lysed
in RIPA buffer (50 mM Tris,pH 8.0, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1%
85
SDS) containing protease and phosphatase inhibitors, plus SDS sample buffer (2% SDS, 0.6M
DTT, 62.5mM Tris, 10% glycerol). Equal quantities of neuronal lysates were subjected to
SDS-PAGE and Western blot analysis, and probed with the indicated antibodies at the
concentrations included in table 3.1. Forebrains or hippocampus were dissected and
Dounce homogenized in RIPA buffer. Lysates were spun at 13,000RPM for 15 min, after
which supernatants were removed and analyzed for protein concentration (Bio-Rad
Protein Assay). SDS buffer was added to equal amounts of protein. Immunoblot analysis
of hippocampal or forebrain lysates was performed using 25-200 pg of protein.
Co-immunoprecipitation studies
HEK293T cells were transfected with various constructs using Lipofectamine 2000.
24 hours post-transfection, cells were lysed with IP buffer (0.4% Triton X-100, 200mM
NaCl, 50mM Tris 7.5) containing protease and phosphatase inhibitors. Equal amounts of
lysates were incubated with anti-antibody-conjugated beads (Sigma, Santa Cruz
Biotechnology) in IP buffer overnight, then washed three times in IP buffer.
Immunoprecipitations from PSD fractions were performed using equal amounts of
protein and rocking the lysates with the 1 pg of the indicated antibodies plus IP buffer
overnight at 4*. Protein A or protein G sepharose beads were then added and allowed to
rock with the antibody/protein complexes for -2hrs at 4*. Beads were then washed 3-5
times with IP buffer. Immune complexes were eluted by addition of sample buffer and
boiling and analyzed by SDS-PAGE.
In Vitro kinase-IP assay of GST-ATF4 and GFP-mDISC1
The generation of GST-ATF4 is described previously (Frank et al., 2010). Briefly, bacteria
containg the pGEXT-4T2-WTATF4, which places the mouse ATF4 cDNA under control of a
LacZ promotor was grown to an O.D. of 0.3 before being stimulated to produce ATF4 by 0.1 pM
86
IPTG. The bacteria were lysed and the GST-ATF4 was collected using glutathione conjugated
sepharose beads and then eluted with elution buffer. The protein concentration of the elutate
was determined. .5ug of protein was resuspended in 250 pl PKA kinase reaction buffer
supplemented with 200 pM ATP.
HEK293-T cells were transfected with GFP-mDISC1T1 and lysed 24 hours post transfection in
IP buffer (0.4% Triton X-100, 200mM NaCl, 50mM Tris 7.5) with phosphatase and protease
inhibitors. After spinning down the cellular debris and GFP-DlSC1T1 was isolated by binding to
50pg GFP-agarose beads (B2, Santa Cruz) for 1 hour. The beads were washed 4 times with the
IP buffer, then twice with PBS: for the last wash, the beads were resusupended in 5ml PBS with
protease inhibitor, and 1.2ml of the resuspended PBS-beads were aliquotted into 4 eppendorf
tubes. After the PBS was removed from the GFP-agarose beads, 50 pl GST-ATF4 in PKA
kinase buffer was added to each reaction condition. 2500units of PKA, 2500units of CIP, or .5ug
BSA was added into the reaction mixture and allowed to incubate at room temperature for 7
minutes before washing again 5 times with IP buffer. For the GFP-only input, the beads were
washed after the addition of BSA in PKA kinase reaction buffer without GST-ATF4. 2% input of
GST-ATF4 is shown.
In vitro-transcription- Hot Kinase assay.
3xFLAG empty vector, mDISC1 WT, 710A, 736A, and 744A were subjected to in-vitro
transcription/translation using the TnT Quick Coupled Transcriptoin/Tanslation System
(Promega). Briefly, the system is a reticulocyte lysate based system in which RNA polymerase,
nucelotides, saltes, and RNAsin RNAse inhibitor is combined with the reticulocyte lysate. The
3xFLAG vector can be driven by T7 RNA polymerase. 1 pg of 3xFLAG-mDISC1 was added into
40ul TnT master mix supplemented with 1ul1mM methionine, mixed, spun down at 1000xg for
10 seconds and incubated at 300C for 1 hour. The lysate was then added to 20pg anti-FLAG
(m2)-conjugated agarose beads resuspended in 500pl IP buffer, washed 5 times using IP buffer,
87
then resuspended in 20pl PKA kinase buffer described above supplanted with 2.5 pCi y-32ATP.
The reaction was incubated for 20 minutes at RT, then stopped by placing on ice for 5 min,
followed by the addition of 4 pl 6xSDS-PAGE buffer, and boiling. The samples were subjected to
SDS-PAGE, and subjected to commassie staining to confirm equal loading of mDISC1 protein.
The gel was the dried and subjected to film exposure to ascertain the differential incorporation
of y-32ATP.
Data Analysis
All data was analyzed using Prism software (GraphPad Software, Inc, La Jolla, CA).
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nutrient-sensing response pathway that activates the human asparagine synthetase gene. J Biol
Chem 277:24120-24127.
Taurin S, Sandbo N, Yau DM, Sethakorn N, Dulin NO (2008) Phosphorylation of beta-catenin by
PKA promotes ATP-induced proliferation of vascular smooth muscle cells. American journal of
physiology Cell physiology 294:C1 169-1174.
Zhang HT, Huang Y, Jin SL, Frith SA, Suvarna N, Conti M, O'Donnell JM (2002)
Antidepressant-like profile and reduced sensitivity to rolipram in mice deficient in the PDE4D
phosphodiesterase enzyme. Neuropsychopharmacology 27:587-595.
Zhang J, Herman EH, Robertson DG, Reily MD, Knapton A, Ratajczak HV, Rifai N, Honchel R,
Blanchard KT, Stoll RE, Sistare FD (2006) Mechanisms and biomarkers of cardiovascular injury
induced by phosphodiesterase inhibitor Ill SK&F 95654 in the spontaneously hypertensive rat.
Toxicologic pathology 34:152-163.
Zhang K, Farooqui SM, O'Donnell JM (1999) Ontogeny of rolipram-sensitive, low-K(m), cyclic
AMP-specific phosphodiesterase in rat brain. Brain research Developmental brain research
112:11-19.
92
CHAPTER 3
Investigation of the role of BCL9, a key regulator of canonical Wnt signaling-mediated
transcription, in neuronal development
Takahiro Soda, Konstatinos Meletis, Yea Jin Kaiser-Woo, Omer Durak, Cillian King, Grace
Taylor, Zack Flood, Thomas Rando, Michel Aguet and Li-Huei Tsai.
93
ABSTRACT
BCL9 is a key regulator of Wnt signaling and is required for TCF/LEF transcription. It is also one
of the nine genes in the critical region of the 1q21.1 copy number variant (CNV). A microdeletion
of the 1q21.1 region is associated with schizophrenia and autism, while a microduplication of
the region is associated with macrocephaly and autism. To assess the role of BCL9 in neuronal
development and psychiatric disorder phenotype, we crossed mice with the flank-loxed BCL9
allele to Nestin-CRE transgenic mice, creating mice with a targeted deletion of BCL9 in neuronal
progenitors. To identify novel functions of BCL9 that can account for its transactivational activity,
we conducted a yeast-two-hybrid screen to identify novel interactors.
INTRODUCTION
Copy number variation of the 1q21.1 region has been identified to associate with a
number of neurodevelopmental disorders (2008; Brunetti-Pierri et al., 2008; Mefford et al., 2008;
Stefansson et al., 2008a). The 1q21.1 microdeletion has been associated with microcephaly
and schizophrenia, while the 1q21.1 microduplication has been associated with macrocephaly
and autism, suggesting the presence of a gene, or a set of genes, within this region that are a
necessary part, and a driver, of head and brain size, as well as neuropsychiatric disorder
manifestation.
Many of the causative genetic perturbations that have been identified in familial cases of
microcephaly and macrocephaly perturb genes that are crucial for proper neuronal development.
We were interested in identifying potential regulators of neuronal development within the CNV
for further evaluation. To this end, we focused on the genes present within the critical region of
the CNV and conducted literature investigations to see if any of them were in pathways known
to be crucial for neuronal development. Of the 9 genes in the critical region, PRKAB2, PDIA3P,
FMD5, CHDI L, BCL9, ACP6, GJA5, and GJA8, BCL9 stood out in terms of it's known role in
the canonical Wnt signaling pathway (Kramps et al., 2002; Brembeck et al., 2004).
94
BCL9 and its homologue BCL9L are key components of the canonical Wnt signaling
pathway (Kramps et al., 2002; Adachi et al., 2004; Brembeck et al., 2004). In a baseline state,
cytosolic p-catenin associates with the destruction complex, consisting of Axin, Adenomtous
polyposis coli (APC), dishevelled (DVL), casein kinase a, and Gsk3P (Moon et al., 2004). The
destruction complex phosphorylates multiple residues on p-catenin that facilitate its interaction
to p-TRCP, Skp1-Cullin1-F-box-protein (SCF) E3 ubiquitin ligase complex, which ubiquinates it
and targets it for degradation. Wnt binding to Frizzled and LRP5/6 recruits the destruction
complex to the cell surface, and concurrently Gsk3p activity is repressed by LRP5/6. This leads
to the accumulation of p-catenin in the cytosol, and the recruitment of p-catenin and its binding
partners into the nucleus.
Nueclear p-catenin binds to the TCF/LEF family of transcription factors that are bound to
TCF/LEF binding sites throughout the genome. p-catenin binding to TCF/LEF recruits histone
acetyltransferases such as CBP as well as other co-transcriptional activators, which leads to the
relaxation of the chromatin and the initiation and maintenance of RNA polymerase II (Pol 11)
mediated transcription. BCL9 is known to interact directly with p-catenin (Krieghoff et al., 2006;
Sampietro et al., 2006) and this interaction is crucial for the enhancement of TCF/LEF
transcriptional activity by Wnt stimulation.
BCL9 and its homolog BCL9L share five homology domains, HD1-5. HD1 binds to, and
is essential for the recruitment of Pygopus (Pygol/2), a TCF/LEF transcriptional co-activator to
TCF/LEF binding sites (Hoffmans and Basler, 2007). HD2 has been shown to bind directly to
catenin. BCL9 appears to aid in the retention of p-catenin within the nucleus, indicating that it
may maintain the p-catenin level necessary for transcriptional activity. HD1 and HD2 also
appear to be necessary for BCL9's function in the facilitation of p-catenin-mediated TCF/LEF
transcription. While the function(s) of HD3-5 are yet unknown, a fragment that contains the
95
p-
region between HD4 and HD5 was shown to have transactivation activity (Sustmann et al.,
2008).
A crystal structure has been made that analyzed the Pygo-BCL9 interaction with
methylated histone H3K4 (Fiedler et al., 2008), as well as the p-catenin-BCL9-TCF4 complex
(Sampietro et al., 2006). These complexes seem to be quite discrete, and this has prompted an
interest in BCL9/BCL9L as molecular targets for novel therapeutics. Presumably, modulation of
BCL9's interaction with p-catenin, Pygo, and the histones may be a mechanism to target Wntmediated transcription in a manner that is more specific than targeting Gsk3P or other upstream
kinases.
The known roles of BCL9 in the Wnt pathway lead us to hypothesize that BCL9 may play
a role in the microcephaly/macrocephaly observed in 1q21.1 CNV deletions and duplications. It
also led us to hypothesize that the perturbation of BCL9 levels may recapitulate some of the
phenotypes observed in schizophrenia and autism. To assess the role of a reduction of BCL9
levels specifically in neurodevelopment, we set out to create a conditional mutant of BCL9. We
also hypothesized that BCL9 may interact with other genes that are either implicated in neuronal
development or psychiatric disorders, or open up new avenues for exploration. To do this, we
conducted both biased and unbiased binding assays of BCL9 protein fragments and have
started to characterize a BCL9 conditional knockout mouse.
METHODS AND PRELIMINARY DATA
Generation of a conditional BCL9 mouse
We followed the steps listed in the recombineering website, adapted from Liu and
colleagues (Figure 1) (Liu et al., 2003). Briefly, we decided to add flanking loxP (flox) sites
around exon 2 of BCL9, as the resulting transcript, even if transcribed consisting of exons 1-3
would result in a stop codon:
96
Exon 1, exon 2, exon 3 of mouse BCL9- translated
MetHPSNPKVRSSPSGNTQSSPKSKQEVMVRPPTVMSPSGNPQL
DSKFSNQGKPGGSASQSQPSPCDSKSGGHTPKALPGPGGSMGL
KNGAGNGAKGKGKRERSISADSFDQRDPGTPNDDSDIKECNSAD
H I KSQESQHTPHS MTPSTATAPRSSTPSHGQT PAPE P I SAQKTPA
KVVYVFSTEMAN
Exon1, exon3 predicted translation
Met H P S N P K V R S S P S G N T Q R M Stop
BAC constructs RP23-242D21 and RP23-134D1 were ordered from the bacpac at CHORI
(Frengen et al., 2000), and the region containing the first five coding exons of BCL9 were
transformed into the p1253 vector through homologous recombination. Briefly, -400 base pair
fragments flanking these coding exons were amplified out of DNA obtained from RP23-242D21
and RP23-134D1 and chosen for the presence of a single BamHl site. The fragment isolated
that was homologus to the -400bp's upstream of BCL9's first coding exon was amplified using
primers that added a Notl restriction site. The fragment was digested using Notl/BamHl
enzymes, then ligated into the PL253 vector. The -400bp fragment downstream of BCL9's fifth
coding exon was amplified using a 5' primer that added an Xbal site. The fragment was
digested using Xbal and BamHI, then ligated into the PL253 vector that already contained the
first fragment. After verification of proper insertion of both arms, the PL253-ArmA-ArmB vector
was linearized using BamHl and dephosphorylated using calf-intestine phosphatase, then
purified. EL350 cells, that had been previously transformed to contain RP23-242D21 or Rp23134D1, were grown to optimal optical density (0.2-0.35 A)and induced for exo, bet, and gam
recombination enzymes, were then electroporated with 100ng of linearized PL253-ArmA-ArmB.
The bacteria were then plated on ampicillin plates for selection of colonies that had successfully
97
recircularized PL253. Colonies were then sent to sequencing to identify the ones that had
recombined (i.e. recircularized with the BCL9 sequence in the plasmid) instead of religated.
They will be referred to as PL253-242D21 or PL253-134D1. PL253-242D21 and PL253-134D1
were retransformed into electrocompetent DH1OB cells to select against multimeric plasmids.
To insert the first loxP site we utilized a similar recombination approach, this time using
the PL452 vector. This vector contains a floxed Neomycin cassette (Neo). To recombine this
neomycin cassette into the PL253 construct, we created two recombination arms distal to the
second exon of BCL9. We amplified these two recombination arms using primers flanked by
5'EcoRl and 3' Sall, and 5'BamHI/3'Notl, respectively, for the arms proximal and distal to floxed
Neo, and sequentially ligated them into the construct. The flox Neo cassette with BCL9
homology arms C and D was then subsequently isolated from PL452 by digestion with Notl and
EcoRI, dephosphorylated, and recombination was induced by combining 100 ng of the fragment
into PL253-242D21 and PL253-134D1 containing EL350 cells that had been grown to optimal
density and induced for recombination, as described above. Bacteria that had recombined
PL253-242D21/134D1 to include the Neo cassette were isolated by selection on kanamycin
plates. Colonies were isolated and PL253-242D21/134D1 flox neo was purified. Cre-mediated
excision of the neomycin cassete was induced by transforming the DNA back into EL350
bacteria grown in arabinose-supplanted media. The DNA from this was subsequently
sequenced to identify the clones that had incorporated the single LoxP site.
To add the second LoxP site, the PL451 plasmid was used to insert the second loxP site
in a manner similar to that described above, using homology arms E and F that targeted the
region in the intron between the first and second exons. The PL451 plasmid features a Neo
cassette that is flanked by frt sites, which can also act to excise DNA placed in between the
sites. The region downstream of the Neo cassette contains a single LoxP site that will remain
98
after the expression of flippase (flpe), which will result in the excision of frt-flanked regions.
Proper insertion of the second loxP site was also confirmed by sequencing.
The targeting constructs were sent to the MIT transgenic facility for electroporation into
mouse ES cells. Two days after receiving the electroporated ES cells to analyze for
recombination, a paper was published that characterized a targeted deletion of BCL9/9L in
mouse muscle progenitors (Brack et al., 2009) and our efforts were discontinued. We have
established a collaboration with the group that generated the mice, and have subsequently
obtained the line. Rederivation, isolation of the flank-loxed allele from BCL91 flank-lox, and
backcrossing of the line has been in progress for some time, and we have started analyzing the
effects of a targeted deletion of BCL9 in neuronal progenitor cells (Nestin-CRE targeted
excision).
99
Noti
BamHI
I
PL253
I
Xbal
I
TK
RP23- 242D21
Tra nsformation/Recom bi nation
PL452
TK
PL253-242D21
Tra nsformation/Recombination
PL253flox
Neo
EEEEE6--'UE
TK
Excision
PL451
PL253Ioxp
TK
Tra nsformation/Recombi nation
PL253frt
Neoloxp
I
=M
M -P,W,
-,=MM
MP'M
L:le -I
PL253
flOXP
kI
TK
Excision
U-EE~EEE4E I
TK
Figure 1: Schematic of the insertion scheme for flank-lox sites for generation of the
conditional BCL9 knockout mouse. Dark Blue boxes represent BCL9 coding exons.
100
Yeast-2-Hybrid screen to identify novel interactors of BCL9
The interaction of BCL9 with its known binding partners, Pygo and p-catenin, are
relatively well characterized. The exact armadillo repeat within p-catenin required for the
interaction (Kramps et al., 2002), as well as the region within Pygo that binds BCL9 has been
determined (Stadeli and Basler, 2005). As mentioned previously, the region between HD4-5 has
also shown transcription enhancing ability, although its function has yet to be elucidated. To
identify potential mechanisms of transcriptional enhancement, we sought to determine what, if
any, proteins interact with Homology Domains 4, 5 and its connecting segment. We chose to
carry out a yeast-2-hybrid screen to identify potential binding partners targets for further
validation. For this, we implemented the Matchmaker system developed by Clontech. Briefly, we
constructed a fusion protein between the GAL4-DNA binding domain and the BCL9 HD4-5
fragment by cloning the fragment into the pGBKT7 construct. BCL9 HD4-5 was amplified from
mouse cDNA isolated from adult mouse cortex using the primers HD4-5 Forward
(TAGCGAATTC- TATCCCATGCCTCCAGAGCCC) HD4-5 Reverse (TATCGTCGACTGAAAACCCAGGCCCAGGACC) to generate a fragment that corresponds to amino acids 992
to 1270 in mouse BCL9. The fragment and pGBKT7 vector were digested using EcoRI and Sall,
purified, and then ligated. The ligation product was transformed into DH5a chemically
competent cells, then plated onto Luria broth-agar plates with kanamycin (50pg/ml). Several
colonies were picked and sequenced using the T7 primer for verification.
Once the correct insert had been verified, pGBKT7 was transformed into Y2H gold
yeast cells. Transformed yeast were plated on SD/-trp, SD/-trp/X-a-gal and SD/-trp/X-a-gal/AbA
to test for autoactivation. After verifying that autoactivation did not occur, the bait strain was
grown overnight in 50ml SD/-Trp liquid media at 300C and 250rpm overnight. When the OD600
reached 0.8, yeast cells were pelleted by centrifugation at 1 OOg for 5 minutes. The cells were
resuspended in 5ml SD/-trp, and combined with 1ml of embryonic mouse brain library
101
(Clontech) in Y187 yeast strain. Then, 45 ml of 2xYPDA medium with 50ug/ml Kanamycin
(YPDA/Kan) was added to the mixture, placed in a 2L flask, and incubated for 24 hours at 50
rpm. After veryfing the presence of Zygotes, cells were pelleted by centrifugation at 1OOg for 5
minutes. The cells were then resuspended in 0.5x YPDA/Kan. 100 pl of cells were plated onto
control (SD/-Trp, SD/-Leu, SD/-Leu/-Trp (DDO) at 1/10, 1/100, 1/1000, and 1/10,000 dilutions to
evaluate the necessary number of clones to screen. The rest of the cells were plated on fifty-five
150mm DDO/X/A plates. These plates were incubated for 4 days. Viability of the diploids was
calculated to be around 2%. All the blue colonies were then re-streaked on higher stringency
QDO/X/A plates. The plasmids contained within the colonies that remained were then purified
by yeast miniprep, transformed back into DH1OB, and plated on ampicillin plates. Three
colonies were selected from each transformation reaction, grown overnight, and sent for
sequencing. A total of 300 colonies were screened (Table 1).
Cloning of BCL9 fragments for verification of interactors, in vitro work, and rescue.
Because of the relatively large size of BCL9, we set out to identify the roles of various
fragments of BCL9. To achieve this, we generated multiple fragments of human BCL9 tagged to
an N-terminal GFP by the insertion of these fragments into the pEGFP-C1 Vector (Clontech)
(Figure 2). Interestingly, we found that DISC1 binds to a region of BCL9 that binds to Pygopus,
as well as the region of BCL9 that is known to have transactivational activity. This suggests that
DISC1 may have a more direct role in TCF/LEF transcription than it has been currently ascribed.
102
A.
Mapping hBCL9
hBCL9 1423 aa
Re m7-a~3
se 34-37
aa 46.S33n
Re
100e.1oso
es 19-mLe
/
C-term
N-term
HD
i-4
31
al
a
a
R
21
U
5
:31
-
1C)
(~
B.
C.
IP:GFP
FLAGDISCITI
14
A
2
U.U.
IL
'
A
A
A
A
~
FLAGDISCIT2
2
-'
.~.
jjfjgii/
IB: GFP
1B:GFP
IB: FLAG(DISCI)
-
Bc19
z
IB: FLAG
IB: GFP (Bc19)
ActIl
- GFP
Figure 2: BCL9 interacts with DISCI through several regions. A. Schematic for the design
of hBCL9 fragments tagged with GFP. B. Full-length DISC1 shows interaction with BCL9.
Immunoprecipitation (IP) using anti-FLAG (M2) conjugated agarose beads after overexpression
of FLAG-tagged mouse DISC1 T1 or T2 and Full length GFP tagged hBCL9. BCL9 showed
stronger interaction with mouse DISC1 T1 than T2. C. DISC1 interacts with several BCL9
regions. Immunoprecipitation using anti-FLAG (M2) conjugated agarose beads after
overexpression of FLAG-tagged mouse DISC1 T1 and various GFP-tagged BCL9 fragments.
Fragment2, which contains the HD1 domain of BCL9, showed consistent enrichment after
immunoprecipitation.
103
Discussion
The importance of the Wnt signaling cascade in brain development is well established
(Chenn and Walsh, 2003). BCL9 knockdown has been shown in previous studies to greatly
reduce Wnt-mediated increase in TCF/LEF response (de la Roche et al., 2008) and BCL9 is
known to be expressed in the fetal brain (Willis et al., 1998). It is one of the genes in the critical
region of the 1q21.1 CNV that is associated with a increase in the risk for schizophrenia
(International Schizophrenia, 2008; Stefansson et al., 2008b), microcephaly, macrocephaly,
and autism (Brunetti-Pierri et al., 2008; Mefford et al., 2008) that have been replicated multiple
times (Levinson et al., 2011; Grozeva et al., 2012). SNPs within the gene are associated with an
increased risk for schizophrenia in the Han Chinese population (Li et al., 2011 a). These
evidence make it quite likely that BCL9 will play some role in neuronal development; yet to date
no study of its role in the nervous system has been published.
We have created the necessary reagents to conduct a through investigation of the role
of BCL9 in neuronal development as well as its effect on the behavior of adult mice. The
conditional knockout model is preferable to full-body knockouts because of the specificity, and
to viral injection models because of its reproducibility of effect. Moreover, the mice are much
more likely to be amenable for behavioral testing because we can avoid the effects of the loss of
gene function on the rest of the body. We can also test the effects of gene dosage by comparing
Nestin-CRE* BCL9 " "'" x mice to Nestin-CRE*, BCL9 +'flox and Nestin-CRE+ BCL9 +' as well as
Nestin-CRE- mice.
Comparing the phenotype of the Nestin-CRE* BCL9"
"oxmice to those phenotypes seen
in the P-catenin and GSK3P loss of function models because this will allow us to discern which
aspects of the phenotypes observed in the mice are due to TCF/LEF mediated transcription, as
opposed to perturbations in other aspects downstream of these molecules. Our current
104
understanding for the role of BCL9 would indicate that the noncanonical Wnt signaling pathways
would largely remain intact, and processes such as axonal guidance and neuronal migration
would not be expected to be affected by the loss of BCL9, as the role of the noncanonical Wnt
signaling cascade is more established for these functions; however whether this is the case
remains to be seen.
Recently, a SNP in the Chromodomain containing protein 8 (CHD8) gene was identified
to be significantly associated to autism (Neale et al., 2012). CHD8, like BCL9, is known to also
form a tripartite complex with p-catenin and methylated histone H3K4 residues. However, unlike
BCL9 which is required for transactivation, CHD8 acts to inhibit transcription at TCF/LEF sites.
Though the presence of CHD8 in the same complex with BCL9 has not been shown directly, it
is likely that these two proteins are part of the same complex that acts to regulate TCF/LEF
transcription. It is also possible that these two proteins are actively competing to enhance, or
inhibit, transcription from the same genetic regions. These are testable hypothesis and is an
area of active investigation.
Also quite likely to be present in the same complex is transcriptional adaptor 2A
(ADA2A), encoded by TADA2, present in the 17q12 CNV. ADA2A is the namesake of the ADA2-A-containing complex (ATAC/PAF) double histone acetyltransferase (HAT) complex that has
both HAT as well as histone deubiquitination activity, and helps drive the initiation of PollI
mediated transcription (Nagy et al., 2010). It is also known to be required for TCF/LEF mediated
transcription (Yang et al., 2008). Genetic abnormalities in another component of this complex,
CBP/p300 is the cause of Rubenstein-Taybi syndrome, a syndrome that includes mental
retardation and behavioral abnormalities (Petrij et al., 1995). It is worth noting that the top hit
within our Yeast-2-hybrid screen B9451P1 is likely to aid this process, and is an active area of
investigation as well. That perturbations in the genes that encode proteins within the same
105
complex adds to the likelihood that TCF/LEF mediated transcription plays a role in the risk for,
and/or development of psychiatric disorders.
The characterization of the Nestin-CRE BCL9o**'ox mice should inform us how much to
pursue this line of reasoning. It should also provide interesting insights on the functional
relevance of TCF/LEF mediated transcription on the efficacy of antidepressant treatment.
Should antidepressants be ineffective in these mice, for example, it will be insightful both from a
mechanistic as well as therapeutic standpoint, as it may narrow down a more specific pathway,
from the myriad of pathways activated by receptor signaling. As Nestin-CRE BCL9'O'f'~ mice
would be predicted to have aberrant neurogenesis, in addition to dramatically reduced TCF/LEF
mediated transcription in all neurons, a comparison with mice with subgranular zone irradiation
will inform one the role of TCF/LEF mediated transcription in neurons that have already been
born. The role of TCF/LEF transcription in the efficacy of antidepressants can be tested in this
manner. It is also possible to test many other compounds and treatments to ask a similar
question. In summary I believe that this mouse line will be an important reagent to address a
host of questions relevant to psychiatric disorders, pharmacological mechanisms, and basic
molecular neuroscience.
106
Candidate
HD451P1
Repeat?
Y8
HD451P5
y3
TIMP4
Y3
TSPAN7
y2
?
Y2
?
y4
?
y3
?
Y
AHCYL1
Y2
FAM35A,
y2
HD451P6
y?
RAF1
Y2
SPATA2
y2
YLPM1
Y2
Chr, strand,
start, end, span
3 - 12169575
12173350 3776
X + 38305741
38432410
126670
9 +
80448773
80449713 941
2 +
116319295
116320282
988
10 - 93796828
93797837 1010
2 + 116319295
116320279
985
1 + 110362538
110366379
3842
10 +
88940232
88941201 970
7 - 44368022
44368702 681
3 - 12600115
12601055 941
20 - 47954097
47954885 789
14 +
74318208
74358571
40364
Function
Tissue inhibitor of metalloproteinases 4,netrin
domain-containing protein encoded by this gene
is involved in regulation of platelet aggregation
and recruitment and may play role in hormonal
regulation and endometrial tissue remodeling,
highly expressed in heart, endomertium and
brain
tetraspanin 7, cell-surface proteins
characterized by the presence of four
hydrophobic domains. Mediate signal
transduction events that regulates of cell
development, activation, growth and motility.
Cell surface glycoprotein, role in the control of
neurite outgrowth. Complex with integrins. This
gene is associated with X-linked mental
retardation and neuropsychiatric diseases such
as Huntington's chorea, fragile X syndrome and
myotonic dystrophy
S-adenosyl homocysteine hydrolase homolog,
intracellular Ca2+ regulation, binds to IP3
raf proto-oncogene serine/threonine protein
kinase, MAP kinase kinase kinase (MAP3K),
Activates MEKI and MEK2Mutations in this
gene are associated with Noonan syndrome 5
and LEOPARD syndrome 2.
spermatogenesis associated protein 2
The nuclear PP1 interacting protein ZAP3 (ZAP)
putative nucleoside kinase that complexes with
SAM68, CIA, NF110/45, and HNRNP-G.
107
CLSTN2
HD45IP2
HD45IP3
IGF1 R
KLHL1
7
?
?
?
?
AF338195
ANGEL1
ARRDC3
BC036485
HD451P4
CGI-150
C1orf216
CTSB
DDX21
DDX58
EDNRB
FAM153C
FAM171B
3 + 141456983
141457843
861
may modulate calcium mediated posysynptic
signals, Highest levels in GABAergic neurons,
association with episodic memory recall task
difference (snp rs6439886)
15 +
97324315
97325281
Insulin-like growth factor I receptor, tyrosine
kinase, essential for hippocampal growth
NMDAactivity attenuates signaling via receptor
967
13 - 69173683
69212582
38900
10 - 93796792
93797837 1046
10 - 93796793
93797837 1045
X + 64287007
64287852 846
7 - 44367884
44368784 901
1 + 144401069
144402014
946
8 + 43104650
43105475 826
14 - 76324395
76324734 340
5 - 90700350
90701383 1034
3 - 180218821
180219825
1005
17 + 852171
853012 842
1 - 35952269
35953340 1072
8 - 11739597
11742639 3043
10 +
70407363
70413053 5691
9 + 32449381
32450391 1011
13 - 77367625
77368579 955
5 + 177368594
177406666
38073
2 + 187337894
187338703
MRP2, a target of Nurr1, involved in
differentiation of mesolimbic dopamine neurons,
oligodendrocite elongation, neurite outgrowth
(gsk3 dependent), actin binding
TBP-2-like inducible membrane protein,
protein, AF151908
Cathepsin B
RNA helicase 11,found in nucleoli, c-Jun
mediates its nucleolar localization
RIG-1, cell receptor for RNA, regulates IFN
production after virus infection, suppresses RAS
Endothelin receptor type B, GPCR, activates
PIP3 signaling involved in neuronal progenitor
proliferation hirschprung's disease
108
810
FBLN-5
FLJ35848
GAD1
Galc
GNAL
GUCY2C
IP07
IRF2BP2
JMJD7
KBTBD1 1
KCNH1
KDM3B
KIAA0408
KIAA1434
14 - 91405648
91413783
17 - 40105846
40106495 650
2 +
171425125
171425907
783
14 +
87491112
87492095 984
18 +
11842584
11843552 969
12 +
14683720
14690806 7087
11 + 9423818
9424822 1005
1 - 232806734
232807832
1099
15 +
39907588
39916755 9168
8 + 1941403
1942495 1093
1 - 209097623
209098625
1003
5 +
137799299
137800280
982
6 - 127804060
127805118
1059
20 - 5473261
5474287 1027
LYST
1 - 233942980
233961015
18036
Mal2
8 + 120325950
120326928
979
Secreted, extracellular matrix protein
containing anArg-Gly-Asp (RGD) motif and
calcium-binding EGF-like domains.
Glutamic Acid decarboxylase 1.
galactosylceramidase-->Krabbe disease(myelin
loss)
Golfalpha gene.. Candidate for schiz
Importin 7, ERK phosphorylated at SPS NTS
site interacts with it, required for translocation
p53 signaling, reduces apoptosis if
overexpressed after actinomcin D treatment
Has jumonji domain, may have fusion protein
with pplA2
Kelch repeat and BTB domain containing 11
Pottasium channel hEAG related, voltage-gated
non-inactivating delayed rectifier
Lysine-specific demethylase 3B
chediak-higashi syndrome 1, lysosomal
trafficking regulator, May be required for sorting
endosomal resident proteins into late
multivesicular endosomes by a mechanism
involving microtubules
T-cell differentiation protein2, component of lipid
rafts, localizes to endosomal structures beneath
the apical membrane. Required for transcytosis,
an intracellular transport pathway used to
deliver membrane-bound proteins and
exogenous cargos from the basolateral to the
apical surface
109
NAPEPLD
7 - 102528524
102529509
986
PALLD
9 - 14076780
14077475 696
2 - 10497961
10499493 1533
4 - 170056052
170057073
1022
PCDH9
13 - 66698405
66699345 941
NFI-B
ODC1
P14KA
P14KAP1
P14KAP2
PIP4K2A
PLG
PPPR2B
PPWD1
PSD3
PURA
RNF111
SEPT13
22 19392025
19397046 5022
22 18763559
18768572 5014
22 - 20157333
20162351 5019
10 - 22944727
22945751 1025
6 - 161081425
161082351
927
5 - 146221613
146222477
865
5 + 64916572
64917930 1359
8 - 18640974
18641957 984
5 + 139475455
139476417
963
15 +
57174351
57175716 1366
7 - 45729916
45731092 1177
N-acyl-phosphatidylethanolamine-hydrolyzing
phospholipase D
Nuclear factor 1/B, Recognizes and binds the
palindromic sequence 5'TTGGCNNNNNGCCAA-3' Capable of activating
transcription and replication, affects brain
development
ornithine decarboxylase 1,
NCAM, FGF interactor
Protocadherin 9, cadherin-related neuronal
receptor that localizes to synaptic junctions and
is putatively involved in specific neuronal
connections and signal transduction. Has large
exon that encodes 6 cadherin domains and a
transmembrane region. Two alternatively
spliced transcript variants encoding distinct
isoforms have been found for this gene.
phosphatidylinositol (PI) 4-kinase which
catalyzes the first committed step in the
biosynthesis of phosphatidylinositol 4,5bisphosphate. The protein encoded by this gene
is a type Ill enzyme that is not inhibited by
adenosine
(plasminogen)
intron
peptidylprolyl isomerase, found in splicosome
Pleckstrin homology and SEC7 domaincontaining protein 3, guanine nucleotide
exchange factor
DNA binding, PURalpha
Arkadia, amplifies TGF-beta signaling by
degrading SMAD7
110
SET
SLC29A2
SLC38A1
SLC39A5
SMBT2
STAMBP
TNS3
UBE2V2
UFSP2
UTRN
WWOX
tanscript variant
1
ZNF33A
ZNF567
9 + 130494968
130497301
2334
11 - 65886845
65890068 3224
12 - 44867836
44868786 951
12 - 54916711
54916736
10 + 7276076
7276381 306
2+
73931027
73943507
12481
7 - 47281923
47282373 451
8 + 49136759
49137716 958
4 - 186557712
186563979
6268
6 - 144835142
144835656
515
16 +
77233895
77234945
10 +
38386017
38387039
19 +
41902522
41903609
19 +
42975494
42976530
1051
inhibitor-2 of protein phosphatase-2A, Isoform 1
and isoform 2 are potent inhibitors of protein
phosphatase 2A. Isoform 1 and isoform 2 inhibit
EP300/CREBBP and PCAF-mediated
acetvlation of histones (HAT) and nucleosomes,
Scm-related gene containing four mbt domains
2, upregulated in response to valproic acid
STAM binding protein, involved in cell-cycle
progresison
actin remodeling, integrin-tensin-actin
ubiquitin-conjugating enzyme E2 variant 2, Has
no ubiquitin ligase activity on its own. In The
UBE2V2/UBE2N heterodimer. Makes noncanonical poly-Ub chains linked through 'Lys63'. Does not lead to protein degradation by the
proteasome. Mediates transcriptional activation
of target genes. Plays a role in the control of
progress through the cell cycle and
differentiation. Plays a role in the error-free DNA
repair pathway and contributes to the survival of
cells after DNA damage
Thiol protease which recognizes and
hydrolyzes the peptide bond at the C-terminal
Gly of UFM1.
utrophin (homologous to dystrophin),
WW domain-containing oxidoreductase,
Probable oxidoreductase, which acts as a tumor
suppressor.
1023
1088
1037
ZNF573
LOC401397
(LOC401397),
7 - 112544015
transcript
112545845
variant 1, non1831
Y4
coding RNA.
Table 1. BCL9 HD4-5 Yeast-2-Hybrid results
111
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Chapter 4
Discussion, future directions
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Follow-up studies to: DISC1 acts as a co-repressor to inhibitATF4 mediated transcription
at the PDE4D loci
The role of PDE4D9 in particular in the DISC1 knockdown phenotype, as well as the
function of PDE4D9 in the nervous system is an area of active investigation. PDE4D9 has been
shown to interact with p2-adrenergic receptors in cardiac myocytes (De Arcangelis et al., 2009).
We have conducted a yeast-2 hybrid screen in which we identified two novel interactors for the
unique portion of PDE4D9. We have confirmed one of these interactors by immunoprecipitation
studies. Interestingly, this hit it is a protein known to play a role in the degradation of dopamine
D2 receptors. It is hard to believe that this is purely due to serendipity, given the importance of
dopamine D2 receptor signaling in antipsychotic action. A characterization of the effects of
PDE4D9 knockdown on D2 receptor surface expression and regulation, will likely yield
interesting results.
PDE4D9 overexpression, and assaying for similarities in its phenotype relative to the
existing DISC1 mouse models, is one method in which we may ascertain the role of PDE4D9.
However, overexpression of PDE4D9 could have a confounding effect, such as ectopic
subcellular localization of PDE4D9, potentially resulting in a general PDE4D overexpression
phenotype. To control for this, the overexpression of another long-variant of PDE4D (or another
PDE4 member) may also be overexpressed, and the nonoverlapping phenotypes may be an
indication of the variant's unique roles.
Rolipram, as well as DISC1, are known to inhibit both isoforms of PDE4s. Past studies
have reported a decrease in immobility in the forced swim test using acute doses of GSK3p
(Mao et al., 2009), and rolipram (Zhang et al., 2002). Rolipram's antidepressants effects are
diminished in PDE4D loss of function mice (Zhang et al., 2012), while the antipsychotic-like
effects of rolipram are diminished in PDE4B loss of function mice (Siuciak et al., 2007). Synergy
125
between tricyclic antidepressants and rolipram has been demonstrated in rodents (Itoh et al.,
2004, Marchetti et al., 2010). A recent study has also demonstrated that schizophrenic and
depressive phenotypes seen in mice with missense mutations in the DISC1 gene can both be
rescued by GSK3p and PDE4 in a synergistic manner (Lipina et al., 2012). It is interesting to
note that the synergy was not seen in wildtype mice for some assays, including the forced swim
test. We are assaying whether a similar effect can be seen in the wildtype context, as this may
address an issue of applicability to humans, as equivalent DISC1 mutations to the ones used for
this study have not been reported in humans. However, since their mechanism seems to be
dependent on the differential binding affinities of DISC1 to PDE4B and GSK3P, it may turn out
to be that DISC1 SNPs confer differential response profiles to these compounds, and may be
predictive of response to this form of combination treatment. Nonetheless, we still do not know
just how much of this effect is mediated by PDE4D9.
We attempted double-knockdown of DISC1 and PDE4D9 in mouse dentate gyrus.
Unfortunately, the co-localization of the markers for PDE4D9 and DISC1 shRNA viral
transduction was low, making the interpretation of the results difficult (results not shown). We
are currently taking advantage of a system developed in the Sudhof lab (Pang et al., 2010) in
which two different shRNAs are expressed from a single lentiviral construct, tagged with a single
fluorophore (GFP), yielding a virus that can simultaneously knock down DISC1 and PDE4D9.
Cell culture based characterization of the effects of dual-knockdown, and the pharmacological
synergy of the inhibition of GSK3p and PDE4's are ongoing. The construct also leaves room for
the insertion of an overexpression rescue construct, which can be either short hairpin resistant
PDE4D9, or another PDE4 variant (to assay for specificity of effect on decreasing particular
variant versus lowering overall PDE4 levels).
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Discussion regarding approach to assaying the effect of genetic perturbations that
confer risk to neuropsychiatric disorders
Our current framing of these disorders assumes that schizophrenia, bipolar disorder, and
autism represent distinct categories in the domain of genetic risk, and that this genetic risk will
give rise to specific perturbations in biochemical pathways that will bring about these disorders.
These disorders are defined by a constellation of symptoms and the diagnosis itself is a
hypothesis. It presumes that categorizing patients in this manner will help guide treatment,
because the disorders result from different causes. The initial genome-wide association studies
(GWAS) applied the disorder criteria in order to have a workable hypothesis. However, the
ensuing biochemical and molecular studies to uncover the functional effect of the identified
genetic risks and whatever new therapeutic approaches these yield, need not rely on diagnosis.
It is entirely possible that numerous perturbations in different biochemical pathways underlie a
constellation of symptoms that end up being categorized as the same disorder. It is also entirely
possible that these different manifestations arise from the same etiology and what is beneficial
for one diagnosis will be useful for all. Evidence from the GWAS conducted comparing patients
with these disorders to the general population, have revealed that these disorders share
common genetic risk perturbations. The potential impact of uncovering the consequences of the
genetic risk factors that are common to all these disorders seem more important, than focusing
on efforts that seek to identify which genetic risk factors are specific to each disorder.
Genome-wide association studies have yielded two general classes of perturbations that
confer risk for neuropsychiatric disorders; common alleles that contribute a small risk, and
rare alleles that contribute a significant degree of risk. There is much contention within the field
in how much these alleles contribute to the overall prevalence of these disorders, one model
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assuming that the common alleles account for much of the prevalence, and the other assuming
that the rare alleles can account for much of the prevalence. The implication of assuming one
model or the other as the causative reason behind these psychiatric disorders are discussed in
greater detail elsewhere (Moskvina et al., 2009; Ripke et al., 2011; Rodriguez-Murillo et al.,
2012; Sullivan and Psychiatric Genetics, 2012) and is the source of much tension in the field
today. Suffice it to say that the genetic differences driving both hypotheses are statistically
significant and reflect true genetic differences that associate more or less with disorder.
Importantly, for the first time, these true genetic associations can be tested by biologists, to
determine how these differences lead to disorder association. Many of the functions of the
putative transcripts and proteins which these genetic differences are thought to affect, are
themselves not known, and it would certainly be beneficial to attribute some function to these
transcripts and proteins proximal to or within the areas of association.
Although not necessary, it would also be advantageous to have some testable
hypothesis to assess the function of these transcripts of unknown function. The Wnt pathway is
a useful framework in which to think about the pathways not only because of the apparent
convergence of proteins identified, but also because the reagents and techniques, which can be
used to probe the pathway, are well established.
From the viewpoint, "one must carefully choose which perturbation to study, as the
findings (or lack thereof) will determine the rest of one's career', the advantages of choosing to
study a CNV, or a gene within a CNV isthat the effect of the perturbation on the gene dosage is
known and the increase in risk is higher, giving one more confidence that creating a constructvalid model will produce a phenotype. In a deletion, there is one less copy of that gene/region,
and in duplication, there are more copies. Studying the genes in a gain-or loss of function model
has face validity just based on this fact.
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The use of single-gene deletion/overexpression animal models also assumes that
genetic dosage of the gene being studied will result in an observable biological phenomenon in
and of itself. The disadvantage from the trainee's perspective is that there is more than one
gene in a single CNV, and the loss of function of a single gene within the CNV may not yield a
phenotype. Most of the CNV critical regions identified in human patients also have systemic
regions within the mouse genome. An ideal animal model to test the effect of the CNV would be
to generate CNV models in which the systemic region homologous to the critical region has
been deleted or duplicated. Indeed, mice that mimic the 22q. 11, 16p, and 15p CNV's have
already been created (Prescott et al., 2005; Takumi, 2010; Horev et al., 2011). Our lab has
attempted, without success thus far, to create the 1q21.1 CNV deletion mouse. A cross of a
CNV deletion mouse with overexpression of a single gene (preferably using the endogenous
promotor), or of a CNV duplication mouse with a deletion of a single gene, would be extremely
insightful as to the contribution of that gene to the CNV phenotype.
Many of the SNPs identified as conferring risk are in noncoding regions of the genome.
The study of the effect of common SNPs in disorder requires an extra step, which is in
determining what the effect of these SNPs are on the expression of genes near the region. The
advantage of studying these SNPs is that they are common, and obtaining samples that harbor
the risk alleles is relatively simple. Conversely, because these SNPs are so common, it is quite
likely that the chromosomes from each sample will harbor multiple risk-conferring SNPs.
Another issue is the relatively low increase in risk conferred by each risk SNP. However, I
believe the same approach must be taken for these SNPs as the CNVs: engineer these SNPs in
the same identical genetic background, and assess differences at the biochemical level. One
must be patient, thorough, and potentially make multiple SNP mutations, but the increase in risk
is multiplicative. If these SNPs are affecting a common pathway, the addition of multiple risk
alleles will, after a certain threshold, elicit an observable biochemical effect. Having the right
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assays in place will be critical to the analysis of this type of work; a pathway driven approach
can be taken here as well. From a practical standpoint, by biasing the initial mutations to create
onto those genes that encode proteins in a single testable pathway with a highly sensitive assay,
one may reduce the number of mutations needed to be created before an effect can be seen
(and become publishable). Eventually, a repository of psychiatric disorder associated SNPs
generated on a common human background can be created through the work of multiple groups.
For both types of genetic perturbations, it would be beneficial to have access to patient
postmortem brain samples, or patient-derived neurons/neuronal progenitors to be able to assay
transcript levels and further strengthen the validity of these models. This can either be done at
the genome-wide level or by assaying genes nearby the SNPs and contained in, or near the
CNVs. Because patient samples are not of the identical genetic background, a large number of
patient samples will need to be assayed for the results to bear significant meaning.
It is evident that these approaches will take the commitment and effort of a large number
of people collaborating with each other, and involves a lot of trust: though one may be
pessimistic in thinking that such collaborations simply do not happen in biology, one might look
towards the consortium studies conducted by geneticists that yielded the genetic data with
which the field is about to conduct studies. I hope to be able to contribute, along with many
other people, to the understanding of these disorders, and, with hope, to uncover therapeutic
targets that will improve the lives of those that suffer from them.
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